Tumor-selective ctla-4 antagonists

ABSTRACT

Provided herein are recombinant masking proteins and recombinant ligand proteins useful in treating cancer, neurodegenerative disease, and cardiovascular disease. The recombinant masking proteins provided herein may, inter alia, be used as non-covalent masks of antagonists of, for example, cellular growth factors (e.g., TNF) or cell surface proteins (e.g., CTLA-4).

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/020,806, filed Jul. 3, 2014, the content of which is incorporatedherein by reference in its entirety and for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with government support under Grant Nos. R21CA135216 and P30 CA033572 awarded by the National Cancer Institute. TheGovernment has certain rights in the invention.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED AS AN ASCII FILE

The Sequence Listing written in file 48440-530001 WO_ST25.TXT, createdon Jul. 1, 2015, 50,482 bytes, machine format IBM-PC, MS Windowsoperating system, is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Most protein-based targeted therapies currently in use target molecularmechanisms but not disease sites such as monoclonal antibodies whichbind to diseased cells or to T cells (as in the case of ipilimumab andthe related CTLA-4 antagonist tremelimumab). However, engagement ofthese targets in normal tissues gives rise to adverse events withvarious degrees of severity depending on the molecular target. If thetherapeutic agent induces autoimmune phenomena the toxicity not onlyleads to significant morbidity, but also often necessitates theadministration of immunosuppressants (corticosteroids, TNF-a inhibitors)which interfere with therapeutic intent.

There is a substantial, unmet need in the clinic to develop targetedtherapies with reduced side effects and enhanced efficacy. Oftenreferred to as “magic bullets”, monoclonal antibodies (mAbs)preferentially target diseased tissue and are generally better toleratedthan traditional chemotherapy. In most cases, therapeutic mAbs bind toan antigen that is ‘self’ but overexpressed in the tumor, such as theErbb family members (e.g., EGFR, Her2). However, systemic administrationof these mAbs, at therapeutic doses, leads the mAb to engage antigenexpressed on normal tissues, and thus, can lead to serious adverse sideeffects. As an example, the mAbs cetuximab (Erbitux™) and trastuzumab(Herceptin™) that are currently used to treat neck and colon cancers andbreast cancer, also give rise to acneiform skin eruptions,gastrointestinal irritation and cardiotoxicity. Serious side effects dueto off-target effects have been also been observed with other mAbs, suchas efalizumab (Raptiva™). Indeed, the adverse effects of efalizumab intreatment of psoriasis, recently resulted in efalizumab being withdrawnfrom the market due to the development of progressive multifocalleukoencephalopathy. Adverse side effects of mAbs in clinical use reducethe efficacy of these agents, cause additional, substantial costsrelated to monitoring these side effects, and ultimately reducepatients' quality of life. In fact, the psychological aspect of thesevere skin rashes alone has led patients to discontinue cetuximab, aneffective treatment approved for various epithelial malignancies. Thus,there is a need in the art for treatment options which avoid these andother adverse effects. Provided herein are solutions to these and otherproblems in the art.

BRIEF SUMMARY OF THE INVENTION

Accordingly, herein are provided, inter alia, compositions, kits, andmethods for treating diseases using recombinant masking proteins andrecombinant ligand proteins.

Provided herein are protein compositions. In one aspect these arerecombinant masking proteins including two identical masking proteindomains. Each of the masking protein domains includes (1) a maskingdimerizing domain; (2) a ligand-masking binding domain; and (3) acleavable masking linker connecting the ligand-masking binding domain tothe masking dimerizing domain. The masking protein domains are boundtogether.

Also provided herein are pharmaceutical compositions. In one aspect, thepharmaceutical compositions include a pharmaceutically acceptableexcipient, a recombinant masking protein as described herein, includingembodiments thereof, and a recombinant ligand protein as describedherein, including embodiments thereof.

The recombinant protein compositions and pharmaceutical compositions mayalso be included in kits described herein. In one aspect this is a kitthat includes a recombinant masking protein as described herein,including embodiments thereof, and a recombinant ligand protein asdescribed herein, including embodiments thereof.

Provided herein are methods of treating a disease in a subject in needthereof. In one aspect, the method includes administering to a subject atherapeutically effective amount of a recombinant masking protein and arecombinant ligand protein as described herein including embodimentsthereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Predicted mode of action of the masked CTLA-4 antagonistmodified lipocalin2 (m-Lcn2): A) In normal tissues M-Lcn2 is masked bythe recombinant CTLA-4 fragment tethered to it by N-terminal linkage andhence does not bind to T cells but in prostate cancer tissue,tumor-associated MMP9 cleaves the CTLA-4 mask leading to native CTLA-4engagement and activation of tumor-infiltrating lymphocytes (TILs) (thedesign principle can be applied to monovalent or bivalent m-Lcn2 toexploit the avidity gain of such ligands); B) Predicted atomic structureof the bivalent CTLA-4 mask containing an Fc fragment tethered toMMP9-cleavable extensions that consist of CTLA-4-derived peptides thatfit the m-Lcn2 binding pocket.

FIG. 2: Variant m-Lcn2 and mask designs to facilitate tumor targeting:A) To create a bivalent analog, m-Lcn2 is fused to the human IgG₁ Fcdomain; B) To create a bivalent mask, CTLA-4 is fused to the same Fcdomain, but the linker contains a MMP9 site; C) To enhance tumortargeting, an HA scFv may be fused to the C-terminus of the m-Lcn2-Fcconstruct (MMP9 cleavage site may added to enhance engagement TILs attumor sites) and a non-cleavable linker may also be generated; D) Thesame targeting principle may be applied to the CTLA4-Fc mask (pleaserefer to FIG. 1B).

FIG. 3: Characterization of mask prototype complex with m-Lcn2-Fc andCTLA-4-Fc: A) SEC indicates the formation of a complex (orange trace) at11.4 mL; B) SPR traces indicate m-Lcn2-Fc binds with high affinity toCTLA-4-MMP9-Fc (highest concentration is 300 nM). C) SDS-PAGE ofCTLA-4-MMP9-Fc before and after 8 hrs of MMP9 treatment (control is amutated the MMP9 site that was not cleaved by MMP9 (right panel)).

FIG. 4: Proposed mutations in the CTLA-4 mask to facilitate unmaskingand block CTLA-4 receptor binding on cells.

FIG. 5: Representation of non-covalent CTLA4-Fc mask system. CTLA4antagonist prodrug is systemically administered as a 1:1mLCN2-Fc:CTLA4-Fc complex. A Non-covalent CTLA4-Fc MMP mask inhibitsmLCN2-Fc from binding endogenous CTLA4 in normal, healthy tissue. B Oncethe prodrug enters the tumor microenvironment, local proteaseoverexpression (shown here as MMP) activates the prodrug by cleaving oneor both MMP sites on the ligand mask. Avidity and, therefore, affinityare lost between the mask and mLCN2-Fc, permitting dissociation of maskand enabling mLCN2-Fc to bind to CTLA4 within the tumor.

FIG. 6: Binding stoichiometry of mLCN2-Fc and CTLA4-Fc MMP9 is dependenton initial protein concentrations. A High initial protein concentrations(“Fast” mixture) resulted in multiple high-order binding mixtures, asdemonstrated by SEC. Most protein was bound 1:1 when lower initialconcentrations were used (“Slow” mixture). Peaks 1, 2, and 3 of initialB “Fast” mixture and C “Slow” mixture were isolated and reanalyzed bySEC.

FIG. 7: CTLA4-Fc MMP9 inhibits the engagement of mLCN2-Fc and murineT-cells. Murine splenocytes were isolated and cultured in the absence orpresence of tumor supernatant (naïve or primed, respectively). After 24h, 20 jig CTLA4-Fc MMP9 was added in the competition condition.Subsequently 20 jig 647′mLCN2-Fc was added to all treatment wells. Cellswere analyzed by flow cytometry for CD4⁺ (top row), CD8⁺ (bottom), and647′mLCN2-Fc staining.

FIG. 8: CTLA4-Fc MMP9 inhibits mLCN2-Fc-mediated stimulation of IFN-γproduction by T-cells in vitro. Cytokine IFN-γ production in murinesplenocytes co-cultured with mLCN2-Fc or mLCN2-Fc:CTLA4-Fc (complex) wasanalyzed by 0. Splenocytes from OT-I mice were activated by Aphytohaemagglutinin (PHA), B TAC-expressing B16 melanoma cells in vitro,or C in vivo vaccination with adenovirus encoding TAC antigen. Blacksquares represent the number of IFN-γ producing cells per well. Blackbisecting bar is the average per condition. Statistics provided fromone-tail t-test. * p<0.05; ** p<0.01; *** p<0.001.

FIG. 9: CTLA4-Fc MMP9 binds CD80 and CD86 expressed on live cells. Humanlymphoblast Daudi cells, which naturally express CD80 and CD86, wereincubated with 647″CTLA4-Fc MMP9. CTLA4-Fc binding was assessed by flowcytometry.

FIG. 10: SEC analysis of CD86-Fc interactions CTLA4-Fc mask variants.Equimolar solutions of recombinant human CD86-Fc and CTLA4-Fc variantswere analyzed by analytical SEC. Absorbance at 230 nm is reported.

FIG. 11: SEC analysis of mLCN2-Fc interactions CTLA4-Fc mask variants.Equimolar solutions of mLCN2-Fc and CTLA4-Fc variants were analyzed byanalytical SEC. Absorbance at 230 nm is reported.

DETAILED DESCRIPTION OF THE INVENTION

The abbreviations used herein have their conventional meaning within thechemical and biological arts. The chemical structures and formulae setforth herein are constructed according to the standard rules of chemicalvalency known in the chemical arts.

A “cell” as used herein, refers to a cell carrying out metabolic orother functions sufficient to preserve or replicate its genomic DNA. Acell can be identified by well-known methods in the art including, forexample, presence of an intact membrane, staining by a particular dye,ability to produce progeny or, in the case of a gamete, ability tocombine with a second gamete to produce a viable offspring. Cells mayinclude prokaryotic and eukaroytic cells. Prokaryotic cells include butare not limited to bacteria. Eukaryotic cells include but are notlimited to yeast cells and cells derived from plants and animals, forexample mammalian, insect (e.g., spodoptera) and human cells. Cells maybe useful when they are naturally nonadherent or have been treated notto adhere to surfaces, for example by trypsinization.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues,wherein the polymer may optionally be conjugated to a moiety that doesnot consist of amino acids. The terms apply to amino acid polymers inwhich one or more amino acid residue is an artificial chemical mimeticof a corresponding naturally occurring amino acid, as well as tonaturally occurring amino acid polymers and non-naturally occurringamino acid polymer. A “fusion protein” refers to a chimeric proteinencoding two or more separate protein sequences that is recombinantlyexpressed as a single moiety.

The term “peptidyl” and “peptidyl moiety” means a monovalent peptide.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids sequences encode any given amino acid residue. For instance, thecodons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, atevery position where an alanine is specified by a codon, the codon canbe altered to any of the corresponding codons described without alteringthe encoded polypeptide. Such nucleic acid variations are “silentvariations,” which are one species of conservatively modifiedvariations. Every nucleic acid sequence herein which encodes apolypeptide also describes every possible silent variation of thenucleic acid. One of skill will recognize that each codon in a nucleicacid (except AUG, which is ordinarily the only codon for methionine, andTGG, which is ordinarily the only codon for tryptophan) can be modifiedto yield a functionally identical molecule. Accordingly, each silentvariation of a nucleic acid which encodes a polypeptide is implicit ineach described sequence with respect to the expression product, but notwith respect to actual probe sequences.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another: 1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine(Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L),Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (see, e.g., Creighton, Proteins (1984)).

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form, andcomplements thereof. The term “polynucleotide” refers to a linearsequence of nucleotides. The term “nucleotide” typically refers to asingle unit of a polynucleotide, i.e., a monomer. Nucleotides can beribonucleotides, deoxyribonucleotides, or modified versions thereof.Examples of polynucleotides contemplated herein include single anddouble stranded DNA, single and double stranded RNA (including siRNA),and hybrid molecules having mixtures of single and double stranded DNAand RNA. Nucleic acid as used herein also refers nucleic acids that havethe same basic chemical structure as a naturally occurring nucleicacids. Such analogues have modified sugars and/or modified ringsubstituents, but retain the same basic chemical structure as thenaturally occurring nucleic acid. A nucleic acid mimetic refers tochemical compounds that have a structure that is different the generalchemical structure of a nucleic acid, but that functions in a mannersimilar to a naturally occurring nucleic acid. Examples of suchanalogues include, without limitation, phosphorothiolates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,2-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs).

“Synthetic mRNA” as used herein refers to any mRNA derived throughnon-natural means such as standard oligonucleotide synthesis techniquesor cloning techniques. Such mRNA may also include non-proteinogenicderivatives of naturally occurring nucleotides. Additionally, “syntheticmRNA” herein also includes mRNA that has been expressed throughrecombinant techniques or exogenously, using any expression vehicle,including but not limited to prokaryotic cells, eukaryotic cell lines,and viral methods. “Synthetic mRNA” includes such mRNA that has beenpurified or otherwise obtained from an expression vehicle or system.

The term “modulation”, “modulate”, or “modulator” are used in accordancewith their plain ordinary meaning and refer to the act of changing orvarying one or more properties. “Modulator” refers to a composition thatincreases or decreases the level of a target molecule or the function ofa target molecule or the physical state of the target of the molecule.“Modulation” refers to the process of changing or varying one or moreproperties. For example, as applied to the effects of a modulator on abiological target, to modulate means to change by increasing ordecreasing a property or function of the biological target or the amountof the biological target.

As defined herein, the term “inhibition”, “inhibit”, “inhibiting” andthe like in reference to a protein-inhibitor (e.g. antagonist)interaction means negatively affecting (e.g. decreasing) the activity orfunction of the protein relative to the activity or function of theprotein in the absence of the inhibitor. In embodiments inhibitionrefers to reduction of a disease or symptoms of disease. Thus, inembodiments, inhibition includes, at least in part, partially or totallyblocking stimulation, decreasing, preventing, or delaying activation, orinactivating, desensitizing, or down-regulating signal transduction orenzymatic activity or the amount of a protein.

As defined herein, the term “activation”, “activate”, “activating” andthe like in reference to a protein-activator (e.g. agonist) interactionmeans positively affecting (e.g. increasing) the activity or function ofthe relative to the activity or function of the protein in the absenceof the activator (e.g. composition described herein). Thus, inembodiments, activation may include, at least in part, partially ortotally increasing stimulation, increasing or enabling activation, oractivating, sensitizing, or up-regulating signal transduction orenzymatic activity or the amount of a protein decreased in a disease.

The term “recombinant” when used with reference, for example, to a cell,a nucleic acid, a protein, or a vector, indicates that the cell, nucleicacid, protein or vector has been modified by or is the result oflaboratory methods. Thus, for example, recombinant proteins includeproteins produced by laboratory methods. Recombinant proteins caninclude amino acid residues not found within the native(non-recombinant) form of the protein or can be include amino acidresidues that have been modified, e.g., labeled.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

“Antibody” refers to a polypeptide comprising a framework region from animmunoglobulin gene or fragments thereof that specifically binds andrecognizes an antigen. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.Typically, the antigen-binding region of an antibody will be mostcritical in specificity and affinity of binding. In some embodiments,antibodies or fragments of antibodies may be derived from differentorganisms, including humans, mice, rats, hamsters, camels, etc.Antibodies of the invention may include antibodies that have beenmodified or mutated at one or more amino acid positions to improve ormodulate a desired function of the antibody (e.g. glycosylation,expression, antigen recognition, effector functions, antigen binding,specificity, etc.).

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain (VL)and variable heavy chain (VH) refer to these light and heavy chainsrespectively. The Fc (i.e. fragment crystallizable region) is the “base”or “tail” of an immunoglobulin and is typically composed of two heavychains that contribute two or three constant domains depending on theclass of the antibody. By binding to specific proteins the Fc regionensures that each antibody generates an appropriate immune response fora given antigen. The Fc region also binds to various cell receptors,such as Fc receptors, and other immune molecules, such as complementproteins.

Antibodies exist, for example, as intact immunoglobulins or as a numberof well-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′2, a dimer ofFab which itself is a light chain joined to VH-CH1 by a disulfide bond.The F(ab)′2 may be reduced under mild conditions to break the disulfidelinkage in the hinge region, thereby converting the F(ab)′2 dimer intoan Fab′ monomer. The Fab′ monomer is essentially Fab with part of thehinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993). Whilevarious antibody fragments are defined in terms of the digestion of anintact antibody, one of skill will appreciate that such fragments may besynthesized de novo either chemically or by using recombinant DNAmethodology. Thus, the term antibody, as used herein, also includesantibody fragments either produced by the modification of wholeantibodies, or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv) or those identified using phagedisplay libraries (see, e.g., McCafferty et al., Nature 348:552-554(1990)).

A single-chain variable fragment (scFv) is typically a fusion protein ofthe variable regions of the heavy (VH) and light chains (VL) ofimmunoglobulins, connected with a short linker peptide of 10 to about 25amino acids. The linker may usually rich in glycine for flexibility, aswell as serine or threonine for solubility. The linker can eitherconnect the N-terminus of the VH with the C-terminus of the VL, or viceversa.

For preparation of suitable antibodies of the invention and for useaccording to the invention, e.g., recombinant, monoclonal, or polyclonalantibodies, many techniques known in the art can be used (see, e.g.,Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al., ImmunologyToday 4: 72 (1983); Cole et al., pp. 77-96 in Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc. (1985); Coligan, Current Protocols inImmunology (1991); Harlow & Lane, Antibodies, A Laboratory Manual(1988); and Goding, Monoclonal Antibodies: Principles and Practice (2ded. 1986)). The genes encoding the heavy and light chains of an antibodyof interest can be cloned from a cell, e.g., the genes encoding amonoclonal antibody can be cloned from a hybridoma and used to produce arecombinant monoclonal antibody. Gene libraries encoding heavy and lightchains of monoclonal antibodies can also be made from hybridoma orplasma cells. Random combinations of the heavy and light chain geneproducts generate a large pool of antibodies with different antigenicspecificity (see, e.g., Kuby, Immunology (3rd ed. 1997)). Techniques forthe production of single chain antibodies or recombinant antibodies(U.S. Pat. No. 4,946,778, U.S. Pat. No. 4,816,567) can be adapted toproduce antibodies to polypeptides of this invention. Also, transgenicmice, or other organisms such as other mammals, may be used to expresshumanized or human antibodies (see, e.g., U.S. Pat. Nos. 5,545,807;5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, Marks et al.,Bio/Technology 10:779-783 (1992); Lonberg et al., Nature 368:856-859(1994); Morrison, Nature 368:812-13 (1994); Fishwild et al., NatureBiotechnology 14:845-51 (1996); Neuberger, Nature Biotechnology 14:826(1996); and Lonberg & Huszar, Intern. Rev. Immunol. 13:65-93 (1995)).Alternatively, phage display technology can be used to identifyantibodies and heteromeric Fab fragments that specifically bind toselected antigens (see, e.g., McCafferty et al., Nature 348:552-554(1990); Marks et al., Biotechnology 10:779-783 (1992)). Antibodies canalso be made bispecific, i.e., able to recognize two different antigens(see, e.g., WO 93/08829, Traunecker et al., EMBO J. 10:3655-3659 (1991);and Suresh et al., Methods in Enzymology 121:210 (1986)). Antibodies canalso be heteroconjugates, e.g., two covalently joined antibodies, orimmunotoxins (see, e.g., U.S. Pat. No. 4,676,980, WO 91/00360; WO92/200373; and EP 03089).

Methods for humanizing or primatizing non-human antibodies are wellknown in the art (e.g., U.S. Pat. Nos. 4,816,567; 5,530,101; 5,859,205;5,585,089; 5,693,761; 5,693,762; 5,777,085; 6,180,370; 6,210,671; and6,329,511; WO 87/02671; EP Patent Application 0173494; Jones et al.(1986) Nature 321:522; and Verhoyen et al. (1988) Science 239:1534).Humanized antibodies are further described in, e.g., Winter and Milstein(1991) Nature 349:293. Generally, a humanized antibody has one or moreamino acid residues introduced into it from a source which is non-human.These non-human amino acid residues are often referred to as importresidues, which are typically taken from an import variable domain.Humanization can be essentially performed following the method of Winterand co-workers (see, e.g., Morrison et al., PNAS USA, 81:6851-6855(1984), Jones et al., Nature 321:522-525 (1986); Riechmann et al.,Nature 332:323-327 (1988); Morrison and Oi, Adv. Immunol., 44:65-92(1988), Verhoeyen et al., Science 239:1534-1536 (1988) and Presta, Curr.Op. Struct. Biol. 2:593-596 (1992), Padlan, Molec. Immun., 28:489-498(1991); Padlan, Molec. Immun., 31(3):169-217 (1994)), by substitutingrodent CDRs or CDR sequences for the corresponding sequences of a humanantibody. Accordingly, such humanized antibodies are chimeric antibodies(U.S. Pat. No. 4,816,567), wherein substantially less than an intacthuman variable domain has been substituted by the corresponding sequencefrom a non-human species. In practice, humanized antibodies aretypically human antibodies in which some CDR residues and possibly someFR residues are substituted by residues from analogous sites in rodentantibodies. For example, polynucleotides comprising a first sequencecoding for humanized immunoglobulin framework regions and a secondsequence set coding for the desired immunoglobulin complementaritydetermining regions can be produced synthetically or by combiningappropriate cDNA and genomic DNA segments. Human constant region DNAsequences can be isolated in accordance with well known procedures froma variety of human cells.

A “chimeric antibody” is an antibody molecule in which (a) the constantregion, or a portion thereof, is altered, replaced or exchanged so thatthe antigen binding site (variable region) is linked to a constantregion of a different or altered class, effector function and/orspecies, or an entirely different molecule which confers new propertiesto the chimeric antibody, e.g., an enzyme, toxin, hormone, growthfactor, drug, etc.; or (b) the variable region, or a portion thereof, isaltered, replaced or exchanged with a variable region having a differentor altered antigen specificity. The preferred antibodies of, and for useaccording to the invention include humanized and/or chimeric monoclonalantibodies.

Techniques for conjugating therapeutic agents to antibodies are wellknown (see, e.g., Amon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery” inControlled Drug Delivery (2^(nd) Ed.), Robinson et al. (eds.), pp.623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers OfCytotoxic Agents In Cancer Therapy: A Review” in Monoclonal Antibodies'84: Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); and Thorpe et al., “The Preparation And CytotoxicProperties Of Antibody-Toxin Conjugates”, Immunol. Rev., 62:119-58(1982)). As used herein, the term “antibody-drug conjugate” or “ADC”refers to a therapeutic agent conjugated or otherwise covalently boundto an antibody. A “therapeutic agent” as referred to herein, is acomposition useful in treating or preventing a disease such as cancer,neurodegenerative disease, or cardiovascular disease.

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction that is determinativeof the presence of the protein, often in a heterogeneous population ofproteins and other biologics. Thus, under designated immunoassayconditions, the specified antibodies bind to a particular protein atleast two times the background and more typically more than 10 to 100times background. Specific binding to an antibody under such conditionsrequires an antibody that is selected for its specificity for aparticular protein. For example, polyclonal antibodies can be selectedto obtain only a subset of antibodies that are specificallyimmunoreactive with the selected antigen and not with other proteins.This selection may be achieved by subtracting out antibodies thatcross-react with other molecules. A variety of immunoassay formats maybe used to select antibodies specifically immunoreactive with aparticular protein. For example, solid-phase ELISA immunoassays areroutinely used to select antibodies specifically immunoreactive with aprotein (see, e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual(1998) for a description of immunoassay formats and conditions that canbe used to determine specific immunoreactivity).

The term “recombinant masking protein” refers to a recombinantlyexpressed polypeptide capable of binding to, or otherwise exhibiting anaffinity for, a particular type of ligand (e.g. polypeptide,polynucleotide, antibody, cellular component, or tissue) or liganddomain as described herein. When non-covalently bound to the ligand(e.g. ligand domain of a recombinant ligand protein as describedherein), a recombinant masking protein inhibits or otherwise preventsthe activity or binding of the recombinant ligand protein to its cognatereceptor or protein. A recombinant masking protein includes one or moreprotein masking domains having one or more “masking dimerizing domains.”The masking dimerizing domains serve as an anchor or structural supportfor a “ligand-masking binding domain.” The masking dimerizing domainsare pairs of identical proteins (e.g. 1-1 (dimer) or 2-2(tetramer)) and,in embodiments, are antibody Fc domains or regions (e.g. full length orfragments of IgG Fc or IgM Fc). Recombinant masking proteins include oneor more “cleavable masking linkers” as described herein. A recombinantmasking protein further includes one or more ligand-masking bindingdomains which non-covalently binds to, and thereby exhibit affinity for,a cognate ligand domain. The ligand-masking binding domain exhibitssufficient affinity for its cognate ligand domain to prevent theactivity or binding of the ligand domain to a protein or receptor. Theligand-masking binding domain may be a polypeptide, polynucleotide,antibody, glycoprotein, or receptor capable of selectively recognizingand binding to a ligand domain.

A “cellular protein domain” is a protein (e.g. full length or functionalor antigenic fragment thereof) having intracellular or extracellularactivity. A cellular protein domain may be associated with a proteinsignal transduction pathway or receptor/ligand binding. A cellularprotein domain may be a cellular growth factor domain (e.g. a fulllength or functional fragment of a protein associated with cellulargrowth) or a cellular surface protein domain (e.g. a full length orfunctional fragment of a protein associated with a protein or receptorlocated on the cell surface). In embodiments, the cellular proteindomain is a protein known to be associated with the cause of, or in theprogression of, a particular disease state (e.g. cancer,neurodegenerative disease, or cardiovascular disease).

The term “recombinant ligand protein” refers to a recombinantlyexpressed polypeptide capable of binding to, or otherwise exhibiting anaffinity for, a particular type of receptor or protein found in or on acell, or ligand-masking binding domain as described herein. Arecombinant ligand protein is composed of one or more “ligand dimerizingdomains” which may serve as an anchor or structural support for a“ligand domain” via attachment through a ligand linker as describedherein. The ligand domain may be a polypeptide, polynucleotide,antibody, glycoprotein, or receptor capable of selectively recognizingand binding to a corresponding ligand-masking binding domain of arecombinant masking protein. The ligand dimerizing domains are pairs ofidentical proteins (e.g. 1-1 (dimer) or 2-2(tetramer)) and, inembodiments, are antibody Fc domains or regions (e.g. full length orfragments of IgG Fc or IgM Fc). The ligand domain may be a cellularprotein binding domain (e.g. a full length or functional fragment of acellular protein recognized or otherwise bound by a particular cellularprotein domain as described herein, including embodiments thereof. Inembodiments, the recombinant ligand protein is an antibody. Inembodiments, the recombinant ligand protein is a monoclonal antibody(mAb). The mAb may be a therapeutic monoclonal antibody. The mAb may bean mAb that recognizes a cellular protein domain as described herein,including embodiments thereof. In embodiments, the mAb is ipilimumab,engineered recombinant lipocalin2, cetuximab, trastuzumab, efalizumab,Etanercept, Adalimumab, Bevacizumab, Gemtuzumab, Infliximab,Natalizumab, Ofatumumab, Panitumumab, Rituximab, Tocilizumab, Abciximab,Ustekinumab, Pertuzumab, or Alemtuzumab.

A “targeting domain” as used herein, refers to a monovalent compositioncapable of binding to, or otherwise exhibiting an affinity for, aparticular type of tissue or component thereof. The addition of atargeting domain to a recombinant masking protein can direct therecombinant masking protein to particular sites within the body.Targeting domains may include, for example, proteins, antibodies,antibody fragments, peptides, carbohydrates, lipids, oligonucleotides,DNA, RNA, or small molecules having a molecular weight less than 2000Daltons. In embodiments, a targeting domain is a single-chain variablefragment (scFv) domain as described herein.

As used herein, “treating” or “treatment of” a condition, disease ordisorder or symptoms associated with a condition, disease or disorderrefers to an approach for obtaining beneficial or desired results,including clinical results. Beneficial or desired clinical results caninclude, but are not limited to, alleviation or amelioration of one ormore symptoms or conditions, diminishment of extent of condition,disorder or disease, stabilization of the state of condition, disorderor disease, prevention of development of condition, disorder or disease,prevention of spread of condition, disorder or disease, delay or slowingof condition, disorder or disease progression, delay or slowing ofcondition, disorder or disease onset, amelioration or palliation of thecondition, disorder or disease state, and remission, whether partial ortotal. “Treating” can also mean prolonging survival of a subject beyondthat expected in the absence of treatment. “Treating” can also meaninhibiting the progression of the condition, disorder or disease,slowing the progression of the condition, disorder or diseasetemporarily, although in some instances, it involves halting theprogression of the condition, disorder or disease permanently.

As used herein the terms treatment, treat, or treating refers to amethod of reducing the effects of one or more symptoms of a disease orcondition characterized by expression of the protease or symptom of thedisease or condition characterized by expression of the protease. Thusin the disclosed method, treatment can refer to a 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of anestablished disease, condition, or symptom of the disease or condition.For example, a method for treating a disease is considered to be atreatment if there is a 10% reduction in one or more symptoms of thedisease in a subject as compared to a control. Thus the reduction can bea 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percentreduction in between 10% and 100% as compared to native or controllevels. It is understood that treatment does not necessarily refer to acure or complete ablation of the disease, condition, or symptoms of thedisease or condition. Further, as used herein, references to decreasing,reducing, or inhibiting include a change of 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90% or greater as compared to a control level and suchterms can include but do not necessarily include complete elimination.

“Contacting” is used in accordance with its plain ordinary meaning andrefers to the process of allowing at least two distinct species (e.g.chemical compounds including biomolecules or cells) to becomesufficiently proximal to react, interact or physically touch. It shouldbe appreciated; however, the resulting reaction product can be produceddirectly from a reaction between the added reagents or from anintermediate from one or more of the added reagents which can beproduced in the reaction mixture.

The term “contacting” may include allowing two species to react,interact, or physically touch, wherein the two species may be, forexample, a cleavable linker as described herein and a protease. Inembodiments contacting includes, for example, allowing a recombinantmasking protein described herein to interact with a recombinant ligandprotein.

As used herein, the term “administering” means oral administration,administration as a suppository, topical contact, intravenous,parenteral, intraperitoneal, intramuscular, intralesional, intrathecal,intranasal or subcutaneous administration, or the implantation of aslow-release device, e.g., a mini-osmotic pump, to a subject.Administration is by any route, including parenteral and transmucosal(e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, ortransdermal). Parenteral administration includes, e.g., intravenous,intramuscular, intra-arteriole, intradermal, subcutaneous,intraperitoneal, intraventricular, and intracranial. Other modes ofdelivery include, but are not limited to, the use of liposomalformulations, intravenous infusion, transdermal patches, etc.

The compound of the invention can be administered alone or can becoadministered to the patient. Coadministration is meant to includesimultaneous or sequential administration of the compound individuallyor in combination (more than one compound or agent). Co-administrationas used herein also refers to administration of ADCs as describedherein. Thus, in embodiments, the preparations can also be combined,when desired, with other active substances (e.g. to reduce metabolicdegradation). The compositions of the present invention can be deliveredtransdermally, by a topical route, formulated as applicator sticks,solutions, suspensions, emulsions, gels, creams, ointments, pastes,jellies, paints, powders, and aerosols. Oral preparations includetablets, pills, powder, dragees, capsules, liquids, lozenges, cachets,gels, syrups, slurries, suspensions, etc., suitable for ingestion by thepatient. Solid form preparations include powders, tablets, pills,capsules, cachets, suppositories, and dispersible granules. Liquid formpreparations include solutions, suspensions, and emulsions, for example,water or water/propylene glycol solutions.

The compositions of the present invention may additionally includecomponents to provide sustained release and/or comfort. Such componentsinclude high molecular weight, anionic mucomimetic polymers, gellingpolysaccharides and finely-divided drug carrier substrates. Thesecomponents are discussed in greater detail in U.S. Pat. Nos. 4,911,920;5,403,841; 5,212,162; and 4,861,760. The entire contents of thesepatents are incorporated herein by reference in their entirety for allpurposes. The compositions of the present invention can also bedelivered as microspheres for slow release in the body. For example,microspheres can be administered via intradermal injection ofdrug-containing microspheres, which slowly release subcutaneously (seeRao, J. Biomater Sci. Polym. Ed. 7:623-645, 1995; as biodegradable andinjectable gel formulations (see, e.g., Gao Pharm. Res. 12:857-863,1995); or, as microspheres for oral administration (see, e.g., Eyles, J.Pharm. Pharmacol. 49:669-674, 1997). In embodiments, the formulations ofthe compositions of the present invention can be delivered by the use ofliposomes which fuse with the cellular membrane or are endocytosed,i.e., by employing receptor ligands attached to the liposome, that bindto surface membrane protein receptors of the cell resulting inendocytosis. By using liposomes, particularly where the liposome surfacecarries receptor ligands specific for target cells, or are otherwisepreferentially directed to a specific organ, one can focus the deliveryof the compositions of the present invention into the target cells invivo. (See, e.g., Al-Muhammed, J. Microencapsul. 13:293-306, 1996;Chonn, Curr. Opin. Biotechnol. 6:698-708, 1995; Ostro, Am. J. Hosp.Pharm. 46:1576-1587, 1989). The compositions of the present inventioncan also be delivered as nanoparticles.

A “effective amount” is an amount sufficient for an active compound toaccomplish a stated purpose relative to the absence of the compound(e.g. achieve the effect for which it is administered, treat a disease,reduce enzyme activity, increase enzyme activity, reduce a signalingpathway, or reduce one or more symptoms of a disease or condition). Anexample of an “effective amount” is an amount sufficient to contributeto the treatment, prevention, or reduction of a symptom or symptoms of adisease, which could also be referred to as a “therapeutically effectiveamount. “A “reduction” of a symptom or symptoms (and grammaticalequivalents of this phrase) means decreasing of the severity orfrequency of the symptom(s), or elimination of the symptom(s). A“prophylactically effective amount” of a drug is an amount of a drugthat, when administered to a subject, will have the intendedprophylactic effect, e.g., preventing or delaying the onset (orreoccurrence) of an injury, disease, pathology or condition, or reducingthe likelihood of the onset (or reoccurrence) of an injury, disease,pathology, or condition, or their symptoms. The full prophylactic effectdoes not necessarily occur by administration of one dose, and may occuronly after administration of a series of doses. Thus, a prophylacticallyeffective amount may be administered in one or more administrations.

Pharmaceutical compositions provided by the present invention includecompositions wherein the active ingredient (e.g. compositions describedherein, including embodiments or examples) is contained in atherapeutically effective amount, i.e., in an amount effective toachieve its intended purpose. The actual amount effective for aparticular application will depend, inter alia, on the condition beingtreated. For any recombinant masking protein and/or recombinant ligandprotein described herein, the therapeutically effective amount can beinitially determined from cell culture assays. Determination of atherapeutically effective amount of a compound of the invention is wellwithin the capabilities of those skilled in the art, especially in lightof the detailed disclosure herein.

The dosage and frequency (single or multiple doses) administered to amammal can vary depending upon a variety of factors, for example,whether the mammal suffers from another disease, and its route ofadministration; size, age, sex, health, body weight, body mass index,and diet of the recipient; nature and extent of symptoms of the diseasebeing treated (e.g. symptoms of cancer, neurodegeneration, orcardiovascular disease and severity of such symptoms), kind ofconcurrent treatment, complications from the disease being treated orother health-related problems. Other therapeutic regimens or agents canbe used in conjunction with the methods and compounds of the invention.Adjustment and manipulation of established dosages (e.g., frequency andduration) are well within the ability of those skilled in the art.

For any compound described herein, the therapeutically effective amountcan be initially determined from cell culture assays. Targetconcentrations will be those concentrations of active compound(s) thatare capable of achieving the methods described herein, as measured usingthe methods described herein or known in the art.

As is well known in the art, therapeutically effective amounts for usein humans can also be determined from animal models. For example, a dosefor humans can be formulated to achieve a concentration that has beenfound to be effective in animals. The dosage in humans can be adjustedby monitoring compounds effectiveness and adjusting the dosage upwardsor downwards, as described above. Adjusting the dose to achieve maximalefficacy in humans based on the methods described above and othermethods is well within the capabilities of the ordinarily skilledartisan.

Dosages may be varied depending upon the requirements of the patient andthe compound being employed. The dose administered to a patient, in thecontext of the present invention should be sufficient to effect abeneficial therapeutic response in the patient over time. The size ofthe dose also will be determined by the existence, nature, and extent ofany adverse side-effects. Determination of the proper dosage for aparticular situation is within the skill of the practitioner. Generally,treatment is initiated with smaller dosages which are less than theoptimum dose of the compound. Thereafter, the dosage is increased bysmall increments until the optimum effect under circumstances isreached.

Dosage amounts and intervals can be adjusted individually to providelevels of the administered compound effective for the particularclinical indication being treated. This will provide a therapeuticregimen that is commensurate with the severity of the individual'sdisease state.

Utilizing the teachings provided herein, an effective prophylactic ortherapeutic treatment regimen can be planned that does not causesubstantial toxicity and yet is effective to treat the clinical symptomsdemonstrated by the particular patient. This planning should involve thecareful choice of active compound by considering factors such ascompound potency, relative bioavailability, patient body weight,presence and severity of adverse side effects, preferred mode ofadministration and the toxicity profile of the selected agent.

“Anti-cancer agent” is used in accordance with its plain ordinarymeaning and refers to a composition (e.g. compound, drug, antagonist,inhibitor, modulator) having antineoplastic properties or the ability toinhibit the growth or proliferation of cells. In embodiments, ananti-cancer agent is a chemotherapeutic. In embodiments, an anti-canceragent is an agent identified herein having utility in methods oftreating cancer. In embodiments, an anti-cancer agent is an agentapproved by the FDA or similar regulatory agency of a country other thanthe USA, for treating cancer.

The compounds described herein can be used in combination with oneanother, with other active agents known to be useful in treating acancer such as anti-cancer agents.

Examples of anti-cancer agents include, but are not limited to, MEK(e.g. MEK1, MEK2, or MEK1 and MEK2) inhibitors (e.g. XL518, CI-1040,PD035901, selumetinib/AZD6244, GSK1120212/trametinib, GDC-0973,ARRY-162, ARRY-300, AZD8330, PD0325901, U0126, PD98059, TAK-733,PD318088, AS703026, BAY 869766), alkylating agents (e.g.,cyclophosphamide, ifosfamide, chlorambucil, busulfan, melphalan,mechlorethamine, uramustine, thiotepa, nitrosoureas, nitrogen mustards(e.g., mechloroethamine, cyclophosphamide, chlorambucil, meiphalan),ethylenimine and methylmelamines (e.g., hexamethlymelamine, thiotepa),alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine,lomusitne, semustine, streptozocin), triazenes (decarbazine)),anti-metabolites (e.g., 5-azathioprine, leucovorin, capecitabine,fludarabine, gemcitabine, pemetrexed, raltitrexed, folic acid analog(e.g., methotrexate), or pyrimidine analogs (e.g., fluorouracil,floxouridine, Cytarabine), purine analogs (e.g., mercaptopurine,thioguanine, pentostatin), etc.), plant alkaloids (e.g., vincristine,vinblastine, vinorelbine, vindesine, podophyllotoxin, paclitaxel,docetaxel, etc.), topoisomerase inhibitors (e.g., irinotecan, topotecan,amsacrine, etoposide (VP16), etoposide phosphate, teniposide, etc.),antitumor antibiotics (e.g., doxorubicin, adriamycin, daunorubicin,epirubicin, actinomycin, bleomycin, mitomycin, mitoxantrone, plicamycin,etc.), platinum-based compounds (e.g. cisplatin, oxaloplatin,carboplatin), anthracenedione (e.g., mitoxantrone), substituted urea(e.g., hydroxyurea), methyl hydrazine derivative (e.g., procarbazine),adrenocortical suppressant (e.g., mitotane, aminoglutethimide),epipodophyllotoxins (e.g., etoposide), antibiotics (e.g., daunorubicin,doxorubicin, bleomycin), enzymes (e.g., L-asparaginase), inhibitors ofmitogen-activated protein kinase signaling (e.g. U0126, PD98059,PD184352, PD0325901, ARRY-142886, SB239063, SP600125, BAY 43-9006,wortmannin, or LY294002, Syk inhibitors, mTOR inhibitors, antibodies(e.g., rituxan), gossyphol, genasense, polyphenol E, Chlorofusin, alltrans-retinoic acid (ATRA), bryostatin, tumor necrosis factor-relatedapoptosis-inducing ligand (TRAIL), 5-aza-2′-deoxycytidine, all transretinoic acid, doxorubicin, vincristine, etoposide, gemcitabine,imatinib (Gleevec®), geldanamycin,17-N-Allylamino-17-Demethoxygeldanamycin (17-AAG), flavopiridol,LY294002, bortezomib, trastuzumab, BAY 11-7082, PKC412, PD184352,20-epi-1, 25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone;aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TKantagonists; altretamine; ambamustine; amidox; amifostine;aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole;andrographolide; angiogenesis inhibitors; antagonist D; antagonist G;antarelix; anti-dorsalizing morphogenetic protein-1; antiandrogen,prostatic carcinoma; antiestrogen; antineoplaston; antisenseoligonucleotides; aphidicolin glycinate; apoptosis gene modulators;apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; argininedeaminase; asulacrine; atamestane; atrimustine; axinastatin 1;axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatinIII derivatives; balanol; batimastat; BCR/ABL antagonists;benzochlorins; benzoylstaurosporine; beta lactam derivatives;beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor;bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistrateneA; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine;calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2;capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRestM3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinaseinhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorins;chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine;clomifene analogues; clotrimazole; collismycin A; collismycin B;combretastatin A4; combretastatin analogue; conagenin; crambescidin 816;crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A;cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate;cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B;deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil;diaziquone; didemnin B; didox; diethylnorspermine;dihydro-5-azacytidine; 9-dioxamycin; diphenyl spiromustine; docosanol;dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA;ebselen; ecomustine; edelfosine; edrecolomab; eflornithine; elemene;emitefur; epirubicin; epristeride; estramustine analogue; estrogenagonists; estrogen antagonists; etanidazole; etoposide phosphate;exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride;flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicinhydrochloride; forfenimex; formestane; fostriecin; fotemustine;gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix;gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam;heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid;idarubicin; idoxifene; idramantone; ilmofosine; ilomastat;imidazoacridones; imiquimod; immunostimulant peptides; insulin-likegrowth factor-1 receptor inhibitor; interferon agonists; interferons;interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact;irsogladine; isobengazole; isohomohalicondrin B; itasetron;jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide;leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole;leukemia inhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine;lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides;maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysininhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone;meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone;miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone;mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growthfactor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonalantibody, human chorionic gonadotrophin; monophosphoryl lipidA+myobacterium cell wall sk; mopidamol; multiple drug resistance geneinhibitor; multiple tumor suppressor 1-based therapy; mustard anticanceragent; mycaperoxide B; mycobacterial cell wall extract; myriaporone;N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin;nemorubicin; neridronic acid; neutral endopeptidase; nilutamide;nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn;O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone;ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin;osaterone; oxaliplatin; oxaunomycin; palauamine; palmitoylrhizoxin;pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine;pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin;pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;propyl bis-acridone; prostaglandin J2; proteasome inhibitors; proteinA-based immune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylerie conjugate; raf antagonists;raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors;ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide;rohitukine; romurtide; roquinimex; rubiginone Bl; ruboxyl; safingol;saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics;semustine; senescence derived inhibitor 1; sense oligonucleotides;signal transduction inhibitors; signal transduction modulators; singlechain antigen-binding protein; sizofuran; sobuzoxane; sodiumborocaptate; sodium phenylacetate; solverol; somatomedin bindingprotein; sonermin; sparfosic acid; spicamycin D; spiromustine;splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-celldivision inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic;thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroidstimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocenebichloride; topsentin; toremifene; totipotent stem cell factor;translation inhibitors; tretinoin; triacetyluridine; triciribine;trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinaseinhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenitalsinus-derived growth inhibitory factor; urokinase receptor antagonists;vapreotide; variolin B; vector system, erythrocyte gene therapy;velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine;vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; zinostatinstimalamer, Adriamycin, Dactinomycin, Bleomycin, Vinblastine, Cisplatin,acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin;aldesleukin; altretamine; ambomycin; ametantrone acetate;aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase;asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa;bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin;bleomycin sulfate; brequinar sodium; bropirimine; busulfan;cactinomycin; calusterone; caracemide; carbetimer; carboplatin;carmustine; carubicin hydrochloride; carzelesin; cedefingol;chlorambucil; cirolemycin; cladribine; crisnatol mesylate;cyclophosphamide; cytarabine; dacarbazine; daunorubicin hydrochloride;decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate;diaziquone; doxorubicin; doxorubicin hydrochloride; droloxifene;droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate;eflornithine hydrochloride; elsamitrucin; enloplatin; enpromate;epipropidine; epirubicin hydrochloride; erbulozole; esorubicinhydrochloride; estramustine; estramustine phosphate sodium; etanidazole;etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride;fazarabine; fenretinide; floxuridine; fludarabine phosphate;fluorouracil; fluorocitabine; fosquidone; fostriecin sodium;gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicinhydrochloride; ifosfamide; iimofosine; interleukin I1 (includingrecombinant interleukin II, or rlL.sub.2), interferon alfa-2a;interferon alfa-2b; interferon alfa-n1; interferon alfa-n3; interferonbeta-1a; interferon gamma-1b; iproplatin; irinotecan hydrochloride;lanreotide acetate; letrozole; leuprolide acetate; liarozolehydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride;masoprocol; maytansine; mechlorethamine hydrochloride; megestrolacetate; melengestrol acetate; melphalan; menogaril; mercaptopurine;methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide;mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper;mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazoie;nogalamycin; ormaplatin; oxisuran; pegaspargase; peliomycin;pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan;piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium;porfiromycin; prednimustine; procarbazine hydrochloride; puromycin;puromycin hydrochloride; pyrazofurin; riboprine; rogletimide; safingol;safingol hydrochloride; semustine; simtrazene; sparfosate sodium;sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin;streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium;tegafur; teloxantrone hydrochloride; temoporfin; teniposide; teroxirone;testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin;tirapazamine; toremifene citrate; trestolone acetate; triciribinephosphate; trimetrexate; trimetrexate glucuronate; triptorelin;tubulozole hydrochloride; uracil mustard; uredepa; vapreotide;verteporfin; vinblastine sulfate; vincristine sulfate; vindesine;vindesine sulfate; vinepidine sulfate; vinglycinate sulfate;vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate;vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicinhydrochloride, agents that arrest cells in the G2-M phases and/ormodulate the formation or stability of microtubules, (e.g. Taxol™ (i.e.paclitaxel), Taxotere™, compounds comprising the taxane skeleton,Erbulozole (i.e. R-55104), Dolastatin 10 (i.e. DLS-10 and NSC-376128),Mivobulin isethionate (i.e. as CI-980), Vincristine, NSC-639829,Discodermolide (i.e. as NVP-XX-A-296), ABT-751 (Abbott, i.e. E-7010),Altorhyrtins (e.g. Altorhyrtin A and Altorhyrtin C), Spongistatins (e.g.Spongistatin 1, Spongistatin 2, Spongistatin 3, Spongistatin 4,Spongistatin 5, Spongistatin 6, Spongistatin 7, Spongistatin 8, andSpongistatin 9), Cemadotin hydrochloride (i.e. LU-103793 andNSC-D-669356), Epothilones (e.g. Epothilone A, Epothilone B, EpothiloneC (i.e. desoxyepothilone A or dEpoA), Epothilone D (i.e. KOS-862, dEpoB,and desoxyepothilone B), Epothilone E, Epothilone F, Epothilone BN-oxide, Epothilone A N-oxide, 16-aza-epothilone B, 21-aminoepothilone B(i.e. BMS-310705), 21-hydroxyepothilone D (i.e. Desoxyepothilone F anddEpoF), 26-fluoroepothilone, Auristatin PE (i.e. NSC-654663), Soblidotin(i.e. TZT-1027), LS-4559-P (Pharmacia, i.e. LS-4577), LS-4578(Pharmacia, i.e. LS-477-P), LS-4477 (Pharmacia), LS-4559 (Pharmacia),RPR-112378 (Aventis), Vincristine sulfate, DZ-3358 (Daiichi), FR-182877(Fujisawa, i.e. WS-9885B), GS-164 (Takeda), GS-198 (Takeda), KAR-2(Hungarian Academy of Sciences), BSF-223651 (BASF, i.e. ILX-651 andLU-223651), SAH-49960 (Lilly/Novartis), SDZ-268970 (Lilly/Novartis),AM-97 (Armad/Kyowa Hakko), AM-132 (Armad), AM-138 (Armad/Kyowa Hakko),IDN-5005 (Indena), Cryptophycin 52 (i.e. LY-355703), AC-7739 (Ajinomoto,i.e. AVE-8063A and CS-39.HCl), AC-7700 (Ajinomoto, i.e. AVE-8062,AVE-8062A, CS-39-L-Ser.HCl, and RPR-258062A), Vitilevuamide, TubulysinA, Canadensol, Centaureidin (i.e. NSC-106969), T-138067 (Tularik, i.e.T-67, TL-138067 and TI-138067), COBRA-1 (Parker Hughes Institute, i.e.DDE-261 and WHI-261), H10 (Kansas State University), H16 (Kansas StateUniversity), Oncocidin A1 (i.e. BTO-956 and DIME), DDE-313 (ParkerHughes Institute), Fijianolide B, Laulimalide, SPA-2 (Parker HughesInstitute), SPA-1 (Parker Hughes Institute, i.e. SPIKET-P), 3-IAABU(Cytoskeleton/Mt. Sinai School of Medicine, i.e. MF-569), Narcosine(also known as NSC-5366), Nascapine, D-24851 (Asta Medica), A-105972(Abbott), Hemiasterlin, 3-BAABU (Cytoskeleton/Mt. Sinai School ofMedicine, i.e. MF-191), TMPN (Arizona State University), Vanadoceneacetylacetonate, T-138026 (Tularik), Monsatrol, lnanocine (i.e.NSC-698666), 3-IAABE (Cytoskeleton/Mt. Sinai School of Medicine),A-204197 (Abbott), T-607 (Tuiarik, i.e. T-900607), RPR-115781 (Aventis),Eleutherobins (such as Desmethyleleutherobin, Desaetyleleutherobin,lsoeleutherobin A, and Z-Eleutherobin), Caribaeoside, Caribaeolin,Halichondrin B, D-64131 (Asta Medica), D-68144 (Asta Medica),Diazonamide A, A-293620 (Abbott), NPI-2350 (Nereus), Taccalonolide A,TUB-245 (Aventis), A-259754 (Abbott), Diozostatin, (−)-Phenylahistin(i.e. NSCL-96F037), D-68838 (Asta Medica), D-68836 (Asta Medica),Myoseverin B, D-43411 (Zentaris, i.e. D-81862), A-289099 (Abbott),A-318315 (Abbott), HTI-286 (i.e. SPA-110, trifluoroacetate salt)(Wyeth), D-82317 (Zentaris), D-82318 (Zentaris), SC-12983 (NCI),Resverastatin phosphate sodium, BPR-OY-007 (National Health ResearchInstitutes), and SSR-250411 (Sanofi)), steroids (e.g., dexamethasone),finasteride, aromatase inhibitors, gonadotropin-releasing hormoneagonists (GnRH) such as goserelin or leuprolide, adrenocorticosteroids(e.g., prednisone), progestins (e.g., hydroxyprogesterone caproate,megestrol acetate, medroxyprogesterone acetate), estrogens (e.g.,diethlystilbestrol, ethinyl estradiol), antiestrogen (e.g., tamoxifen),androgens (e.g., testosterone propionate, fluoxymesterone), antiandrogen(e.g., flutamide), immunostimulants (e.g., Bacillus Calmette-Guérin(BCG), levamisole, interleukin-2, alpha-interferon, etc.), monoclonalantibodies (e.g., anti-CD20, anti-HER2, anti-CD52, anti-HLA-DR, andanti-VEGF monoclonal antibodies), immunotoxins (e.g., anti-CD33monoclonal antibody-calicheamicin conjugate, anti-CD22 monoclonalantibody-pseudomonas exotoxin conjugate, etc.), radioimmunotherapy(e.g., anti-CD20 monoclonal antibody conjugated to ¹¹¹In, ⁹⁰Y, or ¹³¹I,etc.), triptolide, homoharringtonine, dactinomycin, doxorubicin,epirubicin, topotecan, itraconazole, vindesine, cerivastatin,vincristine, deoxyadenosine, sertraline, pitavastatin, irinotecan,clofazimine, 5-nonyloxytryptamine, vemurafenib, dabrafenib, erlotinib,gefitinib, EGFR inhibitors, epidermal growth factor receptor(EGFR)-targeted therapy or therapeutic (e.g. gefitinib (Iressa™),erlotinib (Tarceva™), cetuximab (Erbitux™), lapatinib (Tykerb™),panitumumab (Vectibix™), vandetanib (Caprelsa™), afatinib/BIBW2992,CI-1033/canertinib, neratinib/HKI-272, CP-724714, TAK-285, AST-1306,ARRY334543, ARRY-380, AG-1478, dacomitinib/PF299804, OSI-420/desmethylerlotinib, AZD8931, AEE788, pelitinib/EKB-569, CUDC-101, WZ8040, WZ4002,WZ3146, AG-490, XL647, PD153035, BMS-599626), sorafenib, imatinib,sunitinib, dasatinib, or the like. In embodiments, the compositionsherein may be used in combination with adjunctive agents that may not beeffective alone, but may contribute to the efficacy of the active agentin treating cancer.

The compositions described herein can be used in combination with oneanother, with other active agents known to be useful in treating aneurodegenerative disease. Exemplary active agents include but are notlimited to: levodopa, dopamine agonists (e.g. bromocriptine, pergolide,pramipexole, ropinirole, piribedil, cabergoline, apomorphine, lisuride),MAO-B inhibitors (e.g. selegiline or rasagiline), amantadine,anticholinergics, antipsychotics (e.g. clozapine), cholinesteraseinhibitors, modafinil, or non-steroidal anti-inflammatory drugs. Inembodiments, the compositions herein may be used in combination withadjunctive agents that may not be effective alone, but may contribute tothe efficacy of the active agent in treating neurodegenerative disease.

The compounds described herein can be used in combination with oneanother, with other active agents known to be useful in treating acardiovascular disease such as angiotensin-converting enzyme (ACE)inhibitors, Aldosterone inhibitors, Angiotensin II receptor blockers(ARB), beta blockers, calcium-channel blockers, vasodilators, statins,antiplatelet agents, anticoagulants, or diuretics. In embodiments, thecompositions herein may be used in combination with adjunctive agentsthat may not be effective alone, but may contribute to the efficacy ofthe active agent in treating cardiovascular disease.

In embodiments, co-administration includes administering one activeagent within 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20, or 24 hours of a secondactive agent. Co-administration includes administering two active agentssimultaneously, approximately simultaneously (e.g., within about 1, 5,10, 15, 20, or 30 minutes of each other), or sequentially in any order.In embodiments, co-administration can be accomplished by co-formulation,i.e., preparing a single pharmaceutical composition including bothactive agents. In embodiments, the active agents can be formulatedseparately. In embodiments, the active and/or adjunctive agents may belinked or conjugated to one another.

The term “associated” or “associated with” in the context of a substanceor substance activity or function associated with a disease (e.g. aprotein associated disease, or symptom associated with a cancer,neurodegenerative disease, or cardiovascular disease) means that thedisease (e.g. cancer, neurodegenerative disease, or cardiovasculardisease) is caused or characterized by (in whole or in part) thesubstance or substance activity or function, or a symptom of the diseaseis caused or characterized by (in whole or in part) the substance orsubstance activity or function. As used herein, what is described asbeing associated with a disease, if a causative agent, could be a targetfor treatment of the disease. For example, a disease associated withaberrant CTLA-4 expression or function, may be treated with an agent(e.g. composition as described herein) effective for decreasing thelevel of activity of CTLA-4.

“Control” or “control experiment” is used in accordance with its plainordinary meaning and refers to an experiment in which the subjects orreagents of the experiment are treated as in a parallel experimentexcept for omission of a procedure, reagent, or variable of theexperiment. In some instances, the control is used as a standard ofcomparison in evaluating experimental effects.

“Patient” or “subject in need thereof” refers to a living organismsuffering from or prone to a disease or condition that can be treated byadministration of a composition or pharmaceutical composition asprovided herein. Non-limiting examples include humans, other mammals,bovines, rats, mice, dogs, monkeys, goat, sheep, cows, deer, and othernon-mammalian animals. In some embodiments, a patient is human.

“Disease” or “condition” refer to a state of being or health status of apatient or subject capable of being treated with the compounds ormethods provided herein.

“Pharmaceutically acceptable excipient” and “pharmaceutically acceptablecarrier” refer to a substance that aids the administration of an activeagent to and absorption by a subject and can be included in thecompositions of the present invention without causing a significantadverse toxicological effect on the patient. Non-limiting examples ofpharmaceutically acceptable excipients include water, NaCl, normalsaline solutions, lactated Ringer's, normal sucrose, normal glucose,binders, fillers, disintegrants, lubricants, coatings, sweeteners,flavors, salt solutions (such as Ringer's solution), alcohols, oils,gelatins, carbohydrates such as lactose, amylose or starch, fatty acidesters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, andthe like. Such preparations can be sterilized and, if desired, mixedwith auxiliary agents such as lubricants, preservatives, stabilizers,wetting agents, emulsifiers, salts for influencing osmotic pressure,buffers, coloring, and/or aromatic substances and the like that do notdeleteriously react with the compounds of the invention. One of skill inthe art will recognize that other pharmaceutical excipients are usefulin the present invention.

The term “preparation” is intended to include the formulation of theactive or prodrug form of a composition as provided herein withencapsulating material as a carrier providing a capsule in which theactive component with or without other carriers, is surrounded by acarrier, which is thus in association with it. Similarly, cachets andlozenges are included. Tablets, powders, capsules, pills, cachets, andlozenges can be used as solid dosage forms suitable for oraladministration.

As used herein, the term “cancer” refers to all types of cancer,neoplasm, benign or malignant tumors found in mammals, includingleukemia, carcinomas and sarcomas. Exemplary cancers include cancer ofthe brain, breast, cervix, colon, head & neck, liver, kidney, lung,non-small cell lung, melanoma, mesothelioma, ovary, sarcoma, stomach,uterus and medulloblastoma. Additional examples include, Hodgkin'sDisease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma,ovarian cancer, rhabdomyosarcoma, primary thrombocytosis, primarymacroglobulinemia, primary brain tumors, cancer, malignant pancreaticinsulinoma, malignant carcinoid, urinary bladder cancer, premalignantskin lesions, testicular cancer, lymphomas, thyroid cancer,neuroblastoma, esophageal cancer, genitourinary tract cancer, malignanthypercalcemia, endometrial cancer, adrenal cortical cancer, neoplasms ofthe endocrine and exocrine pancreas, and prostate cancer.

The term “leukemia” refers broadly to progressive, malignant diseases ofthe blood-forming organs and is generally characterized by a distortedproliferation and development of leukocytes and their precursors in theblood and bone marrow. Leukemia is generally clinically classified onthe basis of (1) the duration and character of the disease-acute orchronic; (2) the type of cell involved; myeloid (myelogenous), lymphoid(lymphogenous), or monocytic; and (3) the increase or non-increase inthe number abnormal cells in the blood-leukemic or aleukemic(subleukemic). The P388 leukemia model is widely accepted as beingpredictive of in vivo anti-leukemic activity. It is believed that acompound that tests positive in the P388 assay will generally exhibitsome level of anti-leukemic activity in vivo regardless of the type ofleukemia being treated. Accordingly, the present application includes amethod of treating leukemia, and, preferably, a method of treating acutenonlymphocytic leukemia, chronic lymphocytic leukemia, acutegranulocytic leukemia, chronic granulocytic leukemia, acutepromyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, aleukocythemic leukemia, basophylic leukemia, blast cell leukemia, bovineleukemia, chronic myelocytic leukemia, leukemia cutis, embryonalleukemia, eosinophilic leukemia, Gross' leukemia, hairy-cell leukemia,hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia,stem cell leukemia, acute monocytic leukemia, leukopenic leukemia,lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia,lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia,mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia,monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloidgranulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasmacell leukemia, multiple myeloma, plasmacytic leukemia, promyelocyticleukemia, Rieder cell leukemia, Schilling's leukemia, stem cellleukemia, subleukemic leukemia, and undifferentiated cell leukemia.

The term “sarcoma” generally refers to a tumor which is made up of asubstance like the embryonic connective tissue and is generally composedof closely packed cells embedded in a fibrillar or homogeneoussubstance. Sarcomas which can be treated with a combination ofantineoplastic thiol-binding mitochondrial oxidant and an anticanceragent include a chondrosarcoma, fibrosarcoma, lymphosarcoma,melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma, adiposesarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma,botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma,Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing'ssarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma,granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmentedhemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma,immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma,Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymomasarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma,serocystic sarcoma, synovial sarcoma, and telangiectaltic sarcoma.

The term “melanoma” is taken to mean a tumor arising from themelanocytic system of the skin and other organs. Melanomas which can betreated with a combination of antineoplastic thiol-binding mitochondrialoxidant and an anticancer agent include, for example, acral-lentiginousmelanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman'smelanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma,lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungalmelanoma, and superficial spreading melanoma.

The term “carcinoma” refers to a malignant new growth made up ofepithelial cells tending to infiltrate the surrounding tissues and giverise to metastases. Exemplary carcinomas which can be treated with acombination of antineoplastic thiol-binding mitochondrial oxidant and ananticancer agent include, for example, acinar carcinoma, acinouscarcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinomaadenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolarcell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloidcarcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma,bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma,cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma,comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma encuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cellcarcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma,encephaloid carcinoma, epiermoid carcinoma, carcinoma epithelialeadenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum,gelatiniforni carcinoma, gelatinous carcinoma, giant cell carcinoma,carcinoma gigantocellulare, glandular carcinoma, granulosa cellcarcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellularcarcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypemephroidcarcinoma, infantile embryonal carcinoma, carcinoma in situ,intraepidermal carcinoma, intraepithelial carcinoma, Krompecher'scarcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticularcarcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelialcarcinoma, carcinoma medullare, medullary carcinoma, melanoticcarcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum,carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum,mucous carcinoma, carcinoma myxomatodes, nasopharyngeal carcinoma, oatcell carcinoma, carcinoma ossificans, osteoid carcinoma, papillarycarcinoma, periportal carcinoma, preinvasive carcinoma, prickle cellcarcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reservecell carcinoma, carcinoma sarcomatodes, schneiderian carcinoma,scirrhous carcinoma, carcinoma scroti, signet-ring cell carcinoma,carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidalcell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamouscarcinoma, squamous cell carcinoma, string carcinoma, carcinomatelangiectaticum, carcinoma telangiectodes, transitional cell carcinoma,carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, andcarcinoma villosum.

As used herein, the terms “metastasis,” “metastatic,” and “metastaticcancer” can be used interchangeably and refer to the spread of aproliferative disease or disorder, e.g., cancer, from one organ toanother non-adjacent organ or body part. Cancer occurs at an originatingsite, e.g., breast, which site is referred to as a primary tumor, e.g.,primary breast cancer. Some cancer cells in the primary tumor ororiginating site acquire the ability to penetrate and infiltratesurrounding normal tissue in the local area and/or the ability topenetrate the walls of the lymphatic system or vascular systemcirculating through the system to other sites and tissues in the body. Asecond clinically detectable tumor formed from cancer cells of a primarytumor is referred to as a metastatic or secondary tumor. When cancercells metastasize, the metastatic tumor and its cells are presumed to besimilar to those of the original tumor. Thus, if lung cancermetastasizes to the breast, the secondary tumor at the site of thebreast consists of abnormal lung cells and not abnormal breast cells.The secondary tumor in the breast is referred to a metastatic lungcancer. Thus, the phrase metastatic cancer refers to a disease in whicha subject has or had a primary tumor and has one or more secondarytumors. The phrases non-metastatic cancer or subjects with cancer thatis not metastatic refers to diseases in which subjects have a primarytumor but not one or more secondary tumors. For example, metastatic lungcancer refers to a disease in a subject with or with a history of aprimary lung tumor and with one or more secondary tumors at a secondlocation or multiple locations, e.g., in the breast.

Cancer model organism, as used herein, is an organism exhibiting aphenotype indicative of cancer, or the activity of cancer causingelements, within the organism. The term cancer is defined above. A widevariety of organisms may serve as cancer model organisms, and includefor example, cancer cells and mammalian organisms such as rodents (e.g.mouse or rat) and primates (such as humans).

As used herein, an “autoimmune disease” refers to a disease or disorderthat arises from altered immune reactions by the immune system of asubject, e.g., against substances tissues and/or cells normally presentin the body of the subject. Autoimmune diseases include, but are notlimited to, arthritis, rheumatoid arthritis, psoriatic arthritis,juvenile idiopathic arthritis, scleroderma, systemic scleroderma,multiple sclerosis, systemic lupus erythematosus (SLE), myastheniagravis, juvenile onset diabetes, diabetes mellitus type 1,Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto'sthyroiditis, ankylosing spondylitis, psoriasis, Sjogren's syndrome,vasculitis, glomerulonephritis, auto-immune thyroiditis, Behcet'sdisease, Crohn's disease, ulcerative colitis, bullous pemphigoid,sarcoidosis, psoriasis, ichthyosis, Graves ophthalmopathy, inflammatorybowel disease, Addison's disease, vitiligo, asthma, and allergic asthma.

As used herein, the term “neurodegenerative disease” refers to a diseaseor condition in which the function of a subject's nervous system becomesimpaired. Examples of neurodegenerative diseases that may be treatedwith a compound or method described herein include Alexander's disease,Alper's disease, Alzheimer's disease, Amyotrophic lateral sclerosis,Ataxia telangiectasia, Batten disease (also known asSpielmeyer-Vogt-Sjogren-Batten disease), Bovine spongiformencephalopathy (BSE), Canavan disease, Cockayne syndrome, Corticobasaldegeneration, Creutzfeldt-Jakob disease, frontotemporal dementia,Gerstmann-Sträussler-Scheinker syndrome, Huntington's disease,HIV-associated dementia, Kennedy's disease, Krabbe's disease, kuru, Lewybody dementia, Machado-Joseph disease (Spinocerebellar ataxia type 3),Multiple sclerosis, Multiple System Atrophy, Narcolepsy,Neuroborreliosis, Parkinson's disease, Pelizaeus-Merzbacher Disease,Pick's disease, Primary lateral sclerosis, Prion diseases, Refsum'sdisease, Sandhoffs disease, Schilder's disease, Subacute combineddegeneration of spinal cord secondary to Pernicious Anaemia,Schizophrenia, Spinocerebellar ataxia (multiple types with varyingcharacteristics), Spinal muscular atrophy, Steele-Richardson-Olszewskidisease, Tabes dorsalis, drug-induced Parkinsonism, progressivesupranuclear palsy, corticobasal degeneration, multiple system atrophy,Idiopathic Parkinson's disease, Autosomal dominant Parkinson disease,Parkinson disease, familial, type 1 (PARK1), Parkinson disease 3,autosomal dominant Lewy body (PARK3), Parkinson disease 4, autosomaldominant Lewy body (PARK4), Parkinson disease 5 (PARK5), Parkinsondisease 6, autosomal recessive early-onset (PARK6), Parkinson disease 2,autosomal recessive juvenile (PARK2), Parkinson disease 7, autosomalrecessive early-onset (PARK7), Parkinson disease 8 (PARK8), Parkinsondisease 9 (PARK9), Parkinson disease 10 (PARK10), Parkinson disease 11(PARK11), Parkinson disease 12 (PARK12), or Parkinson disease 13(PARK13).

As used herein, “cardiovascular diseases” refer to diseases associatedwith the heart, blood vessels or both. Cardiovascular diseases include,but are not limited to, coronary heart disease, cardiomyopathy,hypertensive heart disease, heart failure, cardiac dysrhythmias,inflammatory heart disease, peripheral arterial disease, cerebrovasculardisease and inflammatory heart disease.

A “cleavable masking linker” as used herein refers to a portion of apolyvalent linker covalently bonded to a ligand-masking binding domainand covalently bonded to a masking dimerizing domain. In embodiments thecleavable masking linker is recombinantly expressed. In embodiments, thecleavable masking linker is a linker formed by reacting a functional(reactive) group attached to the linker with a masking dimerizing domainusing, for example, conjugate chemistry. In embodiments, the cleavablemasking linker may be a linker formed by reacting a functional(reactive) group attached to the linker with a ligand-masking bindingdomain using, for example, conjugate chemistry. A cleavable maskinglinker may have the formula:

LMBD-CML-MDD  (I).

In Formula (I), LMBD is a ligand-masking binding domain, CML is acleavable masking linker, and MDD is a dimeric masking domain. Asdescribed herein, each recombinant masking protein may include one ormore cleavable masking linkers. A cleavable masking linker includes acleavage site as described herein. FIGS. 1 and 2 set forth exemplaryrecombinant masking proteins having exemplary ligand-masking domains andcleavable masking linkers.

A “ligand linker” as used herein refers to a portion of a polyvalentlinker covalently bonded to a ligand domain and covalently bonded to aligand dimerizing domain. In embodiments the ligand linker isrecombinantly expressed. In embodiments, the ligand linker is a linkerformed by reacting a functional (reactive) group attached to the linkerwith a ligand dimerizing domain using, for example, conjugate chemistry.In embodiments, the ligand linker may be a linker formed by reacting afunctional (reactive) group attached to the linker with the liganddomain using, for example, conjugate chemistry. A ligand linker may havethe formula:

LDD-LL-LD  (II).

In Formula (II), LDD is a ligand dimerizing domain, LL is a ligandlinker, and LD is a ligand domain. As described herein, each recombinantligand protein may include one or more ligand linkers. In embodiments,the ligand linker includes a cleavage site as described herein. FIGS. 1and 2 set forth exemplary recombinant masking proteins having exemplaryligand domains and linker linkers.

A “targeting linker” as used herein refers to a portion of a polyvalentlinker covalently bonded to a targeting domain and covalently bonded toa masking dimerizing domain. In embodiments the targeting linker isrecombinantly expressed. In embodiments, the targeting linker is alinker formed by reacting a functional (reactive) group attached to thelinker with a masking dimerizing domain using, for example, conjugatechemistry. In embodiments, the targeting linker may be a linker formedby reacting a functional (reactive) group attached to the linker with atargeting domain using, for example, conjugate chemistry. A targetinglinker may have the formula:

TD-TL-MDD  (III).

In Formula (III), TD is a targeting domain, TL is a targeting linker,and MDD is a masking dimerization domain. As described herein, eachrecombinant masking protein may include one or more targeting linkersand one or more targeting domains. In embodiments, the targeting linkerincludes a cleavage site as described herein. FIGS. 1 and 2 set forthexemplary recombinant masking proteins having exemplary targetingdomains and targeting linkers.

A “cleavage site” as used herein, refers to a recognizable site forcleavage of a portion of a linker (e.g. polyvalent linker as describedhereinabove) found in a recombinant masking protein or recombinantligand protein described herein. Thus, a cleavage site may be found inthe sequence of a cleavable masking linker, a ligand linker, or atargeting linker as described herein, including embodiments thereof. Inembodiments, the cleavage site is an amino acid sequence that isrecognized and cleaved by a cleavage agent (e.g. a peptidyl sequence).Exemplary cleavage agents include proteins, enzymes, DNAzymes, RNAzymes,metals, acids, and bases. Exemplary cleavage sites are defined herein.

The terms “conjugate” and “conjugate chemistry” refer to reactions withknown reactive groups which proceed under relatively mild conditions.These include, but are not limited to nucleophilic substitutions (e.g.,reactions of amines and alcohols with acyl halides, active esters),electrophilic substitutions (e.g., enamine reactions) and additions tocarbon-carbon and carbon-heteroatom multiple bonds (e.g., Michaelreaction, Diels-Alder addition). These and other useful reactions arediscussed in, for example, March, ADVANCED ORGANIC CHEMISTRY, 3rd Ed.,John Wiley & Sons, New York, 1985; Hermanson, BIOCONJUGATE TECHNIQUES,Academic Press, San Diego, 1996; and Feeney et al., MODIFICATION OFPROTEINS; Advances in Chemistry Series, Vol. 198, American ChemicalSociety, Washington, D.C., 1982.

Useful reactive functional groups used for conjugate chemistries hereininclude, for example:

(a) carboxyl groups and various derivatives thereof including, but notlimited to, N-hydroxysuccinimide esters, N-hydroxybenztriazole esters,acid halides, acyl imidazoles, thioesters, p-nitrophenyl esters, alkyl,alkenyl, alkynyl and aromatic esters;

(b) hydroxyl groups which can be converted to esters, ethers, aldehydes,etc.

(c) haloalkyl groups wherein the halide can be later displaced with anucleophilic group such as, for example, an amine, a carboxylate anion,thiol anion, carbanion, or an alkoxide ion, thereby resulting in thecovalent attachment of a new group at the site of the halogen atom;

(d) dienophile groups which are capable of participating in Diels-Alderreactions such as, for example, maleimido groups;

(e) aldehyde or ketone groups such that subsequent derivatization ispossible via formation of carbonyl derivatives such as, for example,imines, hydrazones, semicarbazones or oximes, or via such mechanisms asGrignard addition or alkyllithium addition;

(f) sulfonyl halide groups for subsequent reaction with amines, forexample, to form sulfonamides;

(g) thiol groups, which can be converted to disulfides, reacted withacyl halides, or bonded to metals such as gold;

(h) amine or sulfhydryl groups, which can be, for example, acylated,alkylated or oxidized;

(i) alkenes, which can undergo, for example, cycloadditions, acylation,Michael addition, etc;

(j) epoxides, which can react with, for example, amines and hydroxylcompounds;

(k) phosphoramidites and other standard functional groups useful innucleic acid synthesis;

(i) metal silicon oxide bonding; and

(l) metal bonding to reactive phosphorus groups (e.g. phosphines) toform, for example, phopshate diester bonds.

The reactive functional groups can be chosen such that they do notparticipate in, or interfere with, the chemical stability of thecompositions described herein. Alternatively, a reactive functionalgroup can be protected from participating in the crosslinking reactionby the presence of a protecting group.

Described herein are ‘antibody prodrugs’ that use tumor-associatedproperties to activate a modified mAb at a disease site. Discoveredherein, a non-covalent recombinant masking protein (e.g. mAb mask), whenintegrated with clinically validated mAbs (i.e. recombinant ligandproteins described herein), reduces the validated mAb's affinity oroccludes its antigen binding site in normal tissue but becomes activatedat the tumor by tumor-specific proteases.

The term “CTLA-4” or “CTLA-4 protein” as provided herein includes any ofthe recombinant or naturally-occurring forms of the cytotoxicT-lymphocyte-associated protein 4 (CTLA-4) or variants or homologsthereof that maintain CTLA-4 protein activity (e.g. within at least 50%,80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to CTLA-4).In some aspects, the variants or homologs have at least 90%, 95%, 96%,97%, 98%, 99% or 100% amino acid sequence identity across the wholesequence or a portion of the sequence (e.g. a 50, 100, 150 or 200continuous amino acid portion) compared to a naturally occurring CTLA-4polypeptide. In embodiments, CTLA-4 is the protein as identified by theNCBI sequence reference GI:83700231, homolog or functional fragmentthereof.

The term “LCN2” as provided herein includes any of the recombinant ornaturally-occurring forms of the lipocalin 2 (LCN2) protein or variantsor homologs thereof that maintain LCN2 protein activity (e.g. within atleast 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity comparedto LCN2). In some aspects, the variants or homologs have at least 90%,95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across thewhole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200continuous amino acid portion) compared to a naturally occurring LCN2polypeptide. In embodiments, the LCN2 protein is the protein asidentified by the NCBI sequence reference GI:38455402, homolog orfunctional fragment thereof.

The modified lipocalin2 (mLcn2) used as a CTLA-4 antagonist (CTLA-4binding protein) herein binds to murine, primate and human CTLA-4. Inembodiments, mLCN2 has the sequence of SEQ ID NO:15. In some aspects,the mLcn2 has at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acidsequence identity across the whole sequence or a portion of the sequence(e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared toSEQ ID NO:15. In embodiments, mLcn2 is the protein as identified by SEQID NO:15 or homolog or functional fragment thereof.

The term “MMP 2” or “MMP 2 protease” as provided herein includes any ofthe recombinant or naturally-occurring forms of the matrixmetalloproteinase 2 (MMP 2) or variants or homologs thereof thatmaintain MMP 2 activity (e.g. within at least 50%, 80%, 90%, 95%, 96%,97%, 98%, 99% or 100% activity compared to MMP 2). In some aspects, thevariants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100%amino acid sequence identity across the whole sequence or a portion ofthe sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion)compared to a naturally occurring MMP 2 polypeptide. In embodiments, MMP2 is the protein as identified by the NCBI sequence referenceGI:189217853, homolog or functional fragment thereof.

The term “MMP 9” or “MMP 9 protease” as provided herein includes any ofthe recombinant or naturally-occurring forms of the matrixmetalloproteinase 9 (MMP 9) or variants or homologs thereof thatmaintain MMP 9 activity (e.g. within at least 50%, 80%, 90%, 95%, 96%,97%, 98%, 99% or 100% activity compared to MMP 9). In some aspects, thevariants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100%amino acid sequence identity across the whole sequence or a portion ofthe sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion)compared to a naturally occurring MMP 9 polypeptide. In embodiments, MMP9 is the protein as identified by the NCBI sequence referenceGI:74272287, homolog or functional fragment thereof.

The term “MMP 13” or “MMP 13 protease” as provided herein includes anyof the recombinant or naturally-occurring forms of the matrixmetalloproteinase 13 (MMP 13) or variants or homologs thereof thatmaintain MMP 13 activity (e.g. within at least 50%, 80%, 90%, 95%, 96%,97%, 98%, 99% or 100% activity compared to MMP 13). In some aspects, thevariants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100%amino acid sequence identity across the whole sequence or a portion ofthe sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion)compared to a naturally occurring MMP 13 polypeptide. In embodiments,MMP 13 is the protein as identified by the NCBI sequence referenceGI:4505209, homolog or functional fragment thereof.

The term “ADAM 9” or “ADAM 9 protease” as provided herein includes anyof the recombinant or naturally-occurring forms of the disintegrin andmetalloprotease domain-containing (ADAM) protein 9 or variants orhomologs thereof that maintain ADAM 9 activity (e.g. within at least50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to ADAM9). In some aspects, the variants or homologs have at least 90%, 95%,96%, 97%, 98%, 99% or 100% amino acid sequence identity across the wholesequence or a portion of the sequence (e.g. a 50, 100, 150 or 200continuous amino acid portion) compared to a naturally occurring ADAM 9polypeptide. In embodiments, ADAM 9 is the protein as identified by theNCBI sequence reference GI:4501915, homolog or functional fragmentthereof.

The term “ADAM 10” or “ADAM 10 protease” as provided herein includes anyof the recombinant or naturally-occurring forms of the disintegrin andmetalloprotease domain-containing (ADAM) protein 10 or variants orhomologs thereof that maintain ADAM 10 activity (e.g. within at least50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to ADAM10). In some aspects, the variants or homologs have at least 90%, 95%,96%, 97%, 98%, 99% or 100% amino acid sequence identity across the wholesequence or a portion of the sequence (e.g. a 50, 100, 150 or 200continuous amino acid portion) compared to a naturally occurring ADAM 10polypeptide. In embodiments, ADAM 10 is the protein as identified by theNCBI sequence reference GI:4557251, homolog or functional fragmentthereof.

The term “ADAM 17” or “ADAM 17 protease” as provided herein includes anyof the recombinant or naturally-occurring forms of the disintegrin andmetalloprotease domain-containing (ADAM) protein 17 or variants orhomologs thereof that maintain ADAM 17 activity (e.g. within at least50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to ADAM17). In some aspects, the variants or homologs have at least 90%, 95%,96%, 97%, 98%, 99% or 100% amino acid sequence identity across the wholesequence or a portion of the sequence (e.g. a 50, 100, 150 or 200continuous amino acid portion) compared to a naturally occurring ADAM 17polypeptide. In embodiments, ADAM 17 is the protein as identified by theNCBI sequence reference GI:73747889, homolog or functional fragmentthereof.

The term “PSA” or “PSA protease” as provided herein includes any of therecombinant or naturally-occurring forms of the prostate-specificantigen (PSA), also known as gamma seminoprotein or kallikrein-3, orvariants or homologs thereof that maintain PSA activity (e.g. within atleast 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity comparedto PSA). In some aspects, the variants or homologs have at least 90%,95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across thewhole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200continuous amino acid portion) compared to a naturally occurring PSApolypeptide. In embodiments, PSA is the protein as identified by theNCBI sequence reference GI:71834853, homolog or functional fragmentthereof.

I. COMPOSITIONS

Provided herein are recombinant masking protein compositions. Therecombinant masking protein includes two identical masking proteindomains. Each masking protein domain includes (1) a masking dimerizingdomain; (2) a ligand-masking binding domain; and (3) a cleavable maskinglinker connecting the ligand-masking binding domain to the maskingdimerizing domain, and where the masking protein domains are boundtogether. The masking protein domains may be bound together through therespective masking dimerizing domains.

The masking dimerizing domain may be an Fc protein domain. The Fcprotein domain may be an IgG or IgM Fc protein. In embodiments, the Fcprotein is an IgG Fc protein. The IgG Fc protein may be an IgG₁ Fcprotein. In embodiments, the IgG₁ Fc protein has a molecular weight ofabout 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45kDa, 50 kDa, 55 kDa, 60 kDa, 70 kDa, 75 kDa, 80 kDa, 85 kDa, 90 kDa, 95kDa, 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, or170 kDa. The IgG₁ Fc protein may have a molecular weight of about 30 kDato about 70 kDa. The IgG₁ Fc protein may have a molecular weight ofabout 40 kDa to about 60 kDa. In embodiments, the Fc protein is an IgMFc protein. The masking dimerizing domain may be a multivalent proteindomain (e.g. preferably dimeric, but also trimeric and tetramericprotein domains). The masking dimerizing domain may be a nanoparticle.

In embodiments, the masking domain is about 50, 75, 100, 125, 150, 175,200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 aminoacids in length. In embodiments, the masking domain is about 50 to about450 amino acids in length. In embodiments, the masking domain is about50 to about 450 amino acids in length. In embodiments, the maskingdomain is about 50 to about 400 amino acids in length. In embodiments,the masking domain is about 50 to about 350 amino acids in length. Inembodiments, the masking domain is about 50 to about 300 amino acids inlength. In embodiments, the masking domain is about 50 to about 250amino acids in length. In embodiments, the masking domain is about 50 toabout 200 amino acids in length. In embodiments, the masking domain isabout 50 to about 150 amino acids in length. In embodiments, the maskingdomain is about 50 to about 100 amino acids in length. In embodiments,the masking domain is about 100 to about 200 amino acids in length. Inembodiments, the masking domain is about 125 to about 200 amino acids inlength. In embodiments, the masking domain is about 150 to about 200amino acids in length. In embodiments, the masking domain is about 175to about 200 amino acids in length.

The ligand-masking binding domain may be a small molecule or a protein(e.g. a cellular protein domain). The cellular protein domain may be acellular growth factor domain, a cellular surface protein domain orfunctional fragment thereof. Thus, in embodiments, the cellular proteindomain may be a cellular growth factor domain. The cellular growthfactor domain may be a cytokine domain. The cellular growth factordomain may be a hormone domain. In embodiments, the cellular growthfactor domain is a PDGF domain (e.g. a full length or functionalfragment of platelet-derived growth factor), an EGF domain (e.g. a fulllength or functional fragment of epidermal growth factor), a TGF domain(e.g. a full length or functional fragment of transforming growth factoralpha or beta), a VEGF domain (e.g. a full length or functional fragmentof vascular endothelial growth factor), a FGF domain (e.g. a full lengthor functional fragment of fibroblast growth factor), or a TNF domain(e.g. a full length or functional fragment of tumor necrosis factor).The cellular growth factor domain may be a TNF domain.

In embodiments, the cellular protein domain is a cellular surfaceprotein domain. The cellular surface protein domain may be a receptortyrosine kinase domain. In embodiments, the receptor tyrosine kinasedomain is a class I, class II, class III, class IV, class V, class VI,class VII, class VII, class IX, class X, class XI, class XII, classXIII, class XIV, class XV, class XVI, or class XVII receptor tyrosinekinase domain (e.g. a full length or functional fragment thereof). Inembodiments, the receptor tyrosine kinase domain is an ErbB receptordomain (e.g. a full length or functional fragment of ErbB). The ErbBdomain may be an EGFR domain (e.g. a full length or functional fragmentof ErbB-1/EGFR), a Her2 domain (e.g. a full length or functionalfragment of ErbB-2/Her2), a Her3 domain (e.g. a full length orfunctional fragment of ErbB-3/Her3), or a Her4 domain (e.g. a fulllength or functional fragment of ErbB-4/Her4). In embodiments, the Erbbdomain is an EGFR domain or a Her2 domain. In embodiments, the receptortyrosine kinase domain is a PDGFR domain (e.g. a full length orfunctional fragment of platelet derived growth factor receptor). Inembodiments, the receptor tyrosine kinase domain is a FGFR domain (e.g.a full length or functional fragment of fibroblast growth factorreceptor). In embodiments, the receptor tyrosine kinase domain is aVEGFR domain (e.g. a full length or functional fragment of vascularendothelial growth factor receptor). In embodiments, the receptortyrosine kinase domain is a HGFR domain (e.g. a full length orfunctional fragment of hepatocyte growth factor receptor). Inembodiments, the cellular surface protein domain is an ErbB receptordomain or a T-cell receptor domain.

The cellular surface protein domain may be a T-cell receptor domain. TheT-cell receptor domain may be a CTLA-4 domain or a functional fragmentthereof. The T-cell receptor domain may be a CTLA-4 domain. Thus, inembodiments, the ligand-masking binding domain is CTLA-4 or a variantthereof. In embodiments, the CTLA-4 domain has the sequence set forth inTable 1.

TABLE 1 SEQ ID NOs of CTLA-4 variants SEQ ID NO: Description SEQ ID NO:1 WT CTLA-4 SEQ ID NO: 2 E31A and R33A CTLA-4 SEQ ID NO: 3 E31S CTLA-4SEQ ID NO: 4 E31K CTLA-4 SEQ ID NO: 5 E31R CTLA-4 SEQ ID NO: 6 E31S,R33E, T51H CTLA-4 SEQ ID NO: 7 T51H CTLA-4 SEQ ID NO: 8 K95A CTLA-4 SEQID NO: 9 E97A CTLA-4 SEQ ID NO: 10 K95A and E97A CTLA-4

In embodiments, the ligand-masking binding domain is an antibody-maskingbinding domain (i.e. a masking biding domain that binds to an antibody).The antibody-masking binding domain may be a monoclonal antibody-maskingdomain (i.e. a masking biding domain that binds to a monoclonalantibody). Thus, in embodiments, the ligand-masking domain is alipocalin 2 masking domain (i.e. a masking binding domain that binds tomLCN2). The ligand-masking domain may be an ipilimumab-masking domain(i.e. a masking biding domain that binds to ipilimumab). Theligand-masking domain may be an efalizumab-masking domain (i.e. amasking biding domain that binds to efalizumab). The ligand-maskingdomain may be a trastuzumab-masking domain (i.e. a masking biding domainthat binds to trastuzumab). The ligand-masking domain may be acetuximab-masking domain (i.e. a masking biding domain that binds tocetuximab). The ligand-masking domain may be an infliximab-maskingdomain (i.e. a masking biding domain that binds to infliximab). Theligand-masking domain may be an etanercept-masking domain (i.e. amasking biding domain that binds to etanercept). The ligand-maskingdomain may be an adalimumab-masking domain (i.e. a masking biding domainthat binds to adalimumab). The ligand-masking domain may be anabciximab-masking domain (i.e. a masking biding domain that binds toabciximab). The ligand-masking domain may be a rituximab-masking domain(i.e. a masking biding domain that binds to rituximab). Theligand-masking domain may be a bevacizumab-masking domain (i.e. amasking biding domain that binds to bevacizumab). The ligand-maskingdomain may be a gemtuzumab-masking domain (i.e. a masking biding domainthat binds to gemtuzumab). The ligand-masking domain may be anatalizumab-masking domain (i.e. a masking biding domain that binds tonatalizumab). The ligand-masking domain may be a panitumumab-maskingdomain (i.e. a masking biding domain that binds to panitumumab). Theligand-masking domain may be an ofatumumab-masking domain (i.e. amasking biding domain that binds to ofatumumab). The ligand-maskingdomain may be a tocilizumab-masking domain (i.e. a masking biding domainthat binds to tocilizumab). The ligand-masking domain may be anustekinumab-masking domain (i.e. a masking biding domain that binds toustekinumab). The ligand-masking domain may be a pertuzumab-maskingdomain (i.e. a masking biding domain that binds to pertuzumab). Theligand-masking domain may be an alemtuzumab-masking domain (i.e. amasking biding domain that binds to alemtuzumab).

The cleavable masking linker may optionally include a protease cleavagesite. In embodiments, the protease cleavage site is recognized by aprotease specific to a cancer or tumor site (i.e. the protease is onlyexpressed or is over-expressed in the cancer or tumor site). Inembodiments, the protease cleavage site is a matrix metalloproteasecleavage site (e.g. a protease cleavage site recognized and cleaved by amatrix metalloprotease as described herein), a disintegrin andmetalloproteinase domain-containing (ADAM) metalloprotease cleavage site(e.g. a protease cleavage site recognized and cleaved by an ADAM) or aprostate specific antigen (PSA) protease cleavage site (e.g. a proteasecleavage site recognized and cleaved by PSA). In embodiments, theprotease cleavage site is a matrix metalloprotease cleavage site. Inembodiments, the protease cleavage site is a site recognized bymetalloproteinase 2 (MMP2) or metalloproteinase 9 (MMP9). Inembodiments, the protease cleavage site is a site recognized bymetalloproteinase 9 (MMP9). In embodiments, the protease cleavage siteis a peptidyl sequence.

In embodiments, the cleavable masking linker includes a enzymaticcleavage site (e.g. a site cleaved via an enzyme-catalyzed reaction), ametal cleavage site (e.g. a site cleaved via a metal-catalyzedreaction), an acid cleave site (e.g. a site cleaved via anacid-catalyzed reaction), a base cleave site (e.g. a site cleaved via abase-catalyzed reaction), or a redox cleavage site (e.g. a site cleavedvia a redox-catalyzed reaction).

The cleavage site on the cleavable masking linker may be cleaved at itscontact point with the ligand-masking binding domain. Alternatively, thecleavage site on the cleavable masking linker may be cleaved at itscontact point with the masking dimerization domain. In embodiments, thecleavage site on the cleavable masking linker may be may be cleaved at apoint between its contact points with the masking dimerization domainand the ligand-masking binding domain.

In embodiments, the recombinant masking protein is bound to arecombinant ligand protein. The recombinant ligand protein includes twoidentical ligand protein domains. Each ligand protein domain includes:(1) a ligand dimerizing domain; (2) a ligand domain; bound to one of theligand-masking binding domains of the recombinant masking protein; and(3) a ligand linker connecting the ligand domain to the liganddimerizing domain, and where the ligand protein domains are boundtogether. The ligand protein domains may be bound together through therespective ligand dimerizing domains.

The ligand dimerizing domain may be an Fc protein domain as describedherein, including embodiments thereof. The ligand dimerizing domain maybe an Fc protein domain that is an IgG₁ Fc protein as described herein,including embodiments thereof.

In embodiments, the recombinant ligand protein is a monoclonal antibody(mAb). In embodiments, the recombinant ligand protein is ipilimumab,lipocalin2, cetuximab, trastuzumab, efalizumab, etanercept, adalimumab,bevacizumab, gemtuzumab, infliximab, natalizumab, ofatumumab,panitumumab, rituximab, tocilizumab, abciximab, ustekinumab, pertuzumab,or alemtuzumab.

The ligand domain may be a cellular protein binding domain. The cellularprotein binding domain may be a cellular growth factor binding domain, acellular surface protein binding domain or functional fragment thereof.Thus, in embodiments, the cellular protein binding domain may be acellular growth factor binding domain. The cellular growth factorbinding domain may be a cytokine binding domain. The cellular growthfactor binding domain may be a hormone binding domain. In embodiments,the cellular growth factor binding domain is a PDGF binding domain (e.g.a full length or functional fragment of platelet-derived growth factor),an EGF binding domain (e.g. a full length or functional fragment ofepidermal growth factor), a TGF binding domain (e.g. a full length orfunctional fragment of transforming growth factor alpha or beta), a VEGFbinding domain (e.g. a full length or functional fragment of vascularendothelial growth factor), a FGF binding domain (e.g. a full length orfunctional fragment of fibroblast growth factor), or a TNF receptordomain (e.g. a full length or functional fragment of tumor necrosisfactor). The cellular growth factor domain may be a TNF receptor domain.

In embodiments, the cellular protein binding domain is a cellularsurface protein binding domain. The cellular surface protein bindingdomain may be a receptor tyrosine kinase binding domain. In embodiments,the receptor tyrosine kinase binding domain is a class I, class II,class III, class IV, class V, class VI, class VII, class VII, class IX,class X, class XI, class XII, class XIII, class XIV, class XV, classXVI, or class XVII receptor tyrosine kinase binding domain (e.g. a fulllength or functional fragment thereof). In embodiments, the receptortyrosine kinase binding domain is an ErbB receptor binding domain (e.g.a full length or functional fragment of ErbB). The ErbB binding domainmay be an EGFR binding domain (e.g. a full length or functional fragmentof ErbB-1/EGFR), a Her2 binding domain (e.g. a full length or functionalfragment of ErbB-2/Her2), a Her3 binding domain (e.g. a full length orfunctional fragment of ErbB-3/Her3), or a Her4 binding domain (e.g. afull length or functional fragment of ErbB-4/Her4). In embodiments, theErbb binding domain is an EGFR binding domain or a Her2 binding domain.In embodiments, the receptor tyrosine kinase binding domain is a PDGFRbinding domain (e.g. a full length or functional fragment of plateletderived growth factor receptor). In embodiments, the receptor tyrosinekinase binding domain is a FGFR binding domain (e.g. a full length orfunctional fragment of fibroblast growth factor receptor). Inembodiments, the receptor tyrosine kinase binding domain is a VEGFRbinding domain (e.g. a full length or functional fragment of vascularendothelial growth factor receptor). In embodiments, the receptortyrosine kinase binding domain is a HGFR binding domain (e.g. a fulllength or functional fragment of hepatocyte growth factor receptor). Inembodiments, the cellular surface protein binding domain is an ErbBreceptor binding domain or a T-cell receptor binding domain.

The cellular surface protein domain may be a T-cell receptor bindingdomain. The T-cell receptor domain may be a CTLA-4 binding domain or afunctional fragment thereof. The T-cell receptor domain may be a CTLA-4binding domain. The CTLA-4 binding domain may be a LCN2 binding domain.

In embodiments, the ligand domain includes a CDR domain. The liganddomain may be an antibody domain.

The ligand linker may optionally include a cleavage site. The ligandlinker cleavage site may include a protease cleavage site, a enzymaticcleavage site, a metal cleavage site, an acid cleave site, a base cleavesite, or a redox cleavage site as described herein, includingembodiments thereof.

In embodiments, the masking dimerizing domains connect to a targetingdomain through a targeting linker. The targeting linker may be connectedto the C-terminus of the masking dimerizing domain. Thus, inembodiments, if the targeting linker is connected to the C-terminus ofthe masking dimerizing domain, the cleavable masking linker may beconnected to the N-terminus of the masking dimerizing domain. Inembodiments, the targeting linker may be connected to the N-terminus ofthe masking dimerizing domain. In such embodiments, the cleavablemasking linker may be connected to the C-terminus of the maskingdimerizing domain. The targeting linker may be a cleavable targetinglinker. In embodiments, the cleavable targeting linker includes aprotease cleavage site. The protease cleavage site is as describedherein, including embodiments thereof. Thus, in embodiments, theprotease cleavage site is a matrix metalloprotease cleavage site, adisintegrin and metalloproteinase domain-containing (ADAM)metalloprotease cleavage site or a prostate specific antigen (PSA)protease cleavage site. In embodiments, the protease cleavage site isdifferent on each masking dimerizing domain. In a preferred embodiment,the protease cleavage site on each targeting linker is cleaved by thesame protease. In embodiments, the protease cleavage site on thetargeting linker is cleaved by the same protease as the proteasecleavage site on the cleavable masking linker.

In embodiments, the cleavable targeting linker includes an enzymaticcleavage site, a metal cleavage site, an acid cleave site, a base cleavesite, or a redox cleavage site as described herein, includingembodiments thereof.

The cleavage site on the cleavable targeting linker may be cleaved atits contact point with the targeting domain. Alternatively, the cleavagesite on the cleavable targeting linker may be cleaved at its contactpoint with the masking dimerization domain. In embodiments, the cleavagesite on the cleavable masking linker may be may be cleaved at a pointbetween its contact points with the masking dimerization domain and thetargeting domain.

The cleavable linkers described herein (e.g. cleavable masking linker,cleavable ligand linker and cleavable targeting linker) may beorthogonal (i.e. one or more sites may be cleaved independently of theother(s) thereby leaving the uncleaved site(s) intact). Thus, inembodiments, the cleavable linkers described herein may be orthogonallycleaved through the same type of catalysis (e.g. protealysis) by usingdifferent cleavage sites or different catalysts (e.g. proteases).Alternatively, the cleavable linkers described herein may beorthogonally cleaved through different types of catalysis.

In embodiments, the targeting domain is a domain that localizes thedomain, and its attached recombinant masking protein to a specific cell,tissue, organ, or location within an organism. In embodiments, thetargeting domain localizes a recombinant masking protein to a cancer(e.g. a cancer cell or tumor). The recombinant masking protein may bebound to a recombinant ligand protein as described herein. The targetingdomain may be a single-chain variable fragment (scFv) domain. Inembodiments, the scFv domain is a hemagglutinin (HA) scFv domain.

In embodiments, the ligand dimerizing domains connect to a targetingdomain through a targeting linker. The targeting linker may be connectedto the C-terminus of the ligand dimerizing domain. Thus, in embodiments,if the targeting linker is connected to the C-terminus of the liganddimerizing domain, the ligand linker may be connected to the N-terminusof the ligand dimerizing domain. In embodiments, the targeting linkermay be connected to the N-terminus of the ligand dimerizing domain. Insuch embodiments, the ligand linker may be connected to the C-terminusof the ligand dimerizing domain. The targeting linker may be a cleavabletargeting linker. The cleavable targeting linker is as described herein,including embodiments thereof. In embodiments, the protease cleavagesite on the targeting linker connecting the targeting domain to theligand dimerization domain is cleaved by the same protease which cleavesthe protease cleavage site on the cleavable masking linker or by thesame protease which cleaves the protease cleavage site on the cleavabletargeting linker connecting a targeting domain to the cleavable maskinglinker. The targeting domain connected to the ligand dimerization is atargeting domain as described herein, including embodiments thereof.Thus, in embodiments, the targeting domain connected to the liganddimerization domain may be a scFv domain. The scFv domain may be ahemaglutinin scFv domain.

II. PHARMACEUTICAL COMPOSITIONS

Provided herein are pharmaceutical compositions. In one aspect, thepharmaceutical compositions include a recombinant masking protein and arecombinant ligand protein as described herein. In another aspect, thepharmaceutical compositions include a recombinant masking protein.

The pharmaceutical composition may be prepared and administered in awide variety of dosage formulations and may be administered orally,rectally, or by injection (e.g. intravenously, intramuscularly,intracutaneously, subcutaneously, intraduodenally, orintraperitoneally).

Solid form preparations include powders, tablets, pills, capsules,cachets, suppositories, and dispersible granules. A solid carrier may beone or more substance that may also act as diluents, flavoring agents,binders, preservatives, tablet disintegrating agents, or anencapsulating material.

In powders, the carrier may be a finely divided solid in a mixture withthe finely divided active component. In tablets, the recombinant proteincompositions described herein may be mixed with the carrier having thenecessary binding properties in suitable proportions and compacted inthe shape and size desired. Suitable carriers are magnesium carbonate,magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch,gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, alow melting wax, cocoa butter, and the like. The term “preparation” isintended to include a formulation of the recombinant proteins describedherein with or without other carriers, and surrounded by a carrier,which is thus in association with it. Similarly, cachets and lozengesare included. Tablets, powders, capsules, pills, cachets, and lozengescan be used as solid dosage forms suitable for oral administration.

For preparing suppositories, a low melting wax, such as a mixture offatty acid glycerides or cocoa butter, is first melted and thecompositions described herein are dispersed homogeneously therein, as bystirring. The molten homogeneous mixture is then poured into convenientsized molds, allowed to cool, and thereby to solidify.

Liquid form preparations include solutions, suspensions, and emulsions,for example, water or water/propylene glycol solutions. For parenteralinjection, liquid preparations can be formulated in solution in aqueouspolyethylene glycol solution.

Aqueous solutions suitable for oral use can be prepared by dissolvingthe active component in water and adding suitable colorants, flavors,stabilizers, and thickening agents as desired. Aqueous suspensionssuitable for oral use can be made by dispersing the finely dividedactive component in water with viscous material, such as natural orsynthetic gums, resins, methylcellulose, sodium carboxymethylcellulose,and other well-known suspending agents.

Also included are solid form preparations that are intended to beconverted, shortly before use, to liquid form preparations for oraladministration. Such liquid forms include solutions, suspensions, andemulsions. These preparations may contain, in addition to thecompositions provided herein, colorants, flavors, stabilizers, buffers,artificial and natural sweeteners, dispersants, thickeners, solubilizingagents, and the like.

The pharmaceutical preparation is preferably in unit dosage form. Insuch form the preparation is subdivided into unit doses containingappropriate quantities of the compositions described herein. The unitdosage form can be a packaged preparation, the package containingdiscrete quantities of preparation, such as packeted tablets, capsules,and powders in vials or ampoules. Also, the unit dosage form can be acapsule, tablet, cachet, or lozenge itself, or it can be the appropriatenumber of any of these in packaged form.

The quantity of active component in a unit dose preparation may bevaried or adjusted according to the particular application and thepotency of the therapeutic agent used. The composition can, if desired,also contain other compatible therapeutic agents.

The pharmaceutical compositions may additionally include components toprovide sustained release and/or comfort. Such components include highmolecular weight, anionic mucomimetic polymers, gelling polysaccharides,and finely-divided drug carrier substrates. These components arediscussed in greater detail in U.S. Pat. Nos. 4,911,920; 5,403,841;5,212,162; and 4,861,760. The entire contents of these patents areincorporated herein by reference in their entirety for all purposes.

The pharmaceutical composition may be intended for intravenous use. Thepharmaceutically acceptable excipient can include buffers to adjust thepH to a desirable range for intravenous use. Many buffers includingsalts of inorganic acids such as phosphate, borate, and sulfate areknown.

The pharmaceutical composition may include compositions wherein thetherapeutic agent is contained in a therapeutically effective amount,i.e., in an amount effective to achieve its intended purpose. The actualamount effective for a particular application will depend, inter alia,on the condition being treated. For example, when administered inmethods to treat cancer, such compositions will contain amounts oftherapeutic agent effective to achieve the desired result.

The dosage and frequency (single or multiple doses) of thepharmaceutical composition administered can vary depending upon avariety of factors, including route of administration; size, age, sex,health, body weight, body mass index, and diet of the recipient; natureand extent of symptoms of the disease being treated; presence of otherdiseases or other health-related problems; kind of concurrent treatment;and complications from any disease or treatment regimen. Othertherapeutic regimens or agents can be used in conjunction with themethods and compounds disclosed herein.

Dosages may be varied depending upon the requirements of the subject andthe compound being employed. The dose administered to a subject, in thecontext of the pharmaceutical compositions presented herein, should besufficient to effect a beneficial therapeutic response in the subjectover time. The size of the dose also will be determined by theexistence, nature, and extent of any adverse side effects. Generally,treatment is initiated with smaller dosages, which are less than theoptimum dose of the compound. Thereafter, the dosage is increased bysmall increments until the optimum effect under circumstances isreached.

Dosage amounts and intervals can be adjusted individually to providelevels of the administered compounds effective for the particularclinical indication being treated. This will provide a therapeuticregimen that is commensurate with the severity of the individual'sdisease state.

Utilizing the teachings provided herein, an effective prophylactic ortherapeutic treatment regimen can be planned that does not causesubstantial toxicity and yet is entirely effective to treat the clinicalsymptoms demonstrated by the particular patient. This planning shouldinvolve the careful choice of therapeutic agent by considering factorssuch as potency, bioavailability, patient body weight, presence andseverity of adverse side effects, preferred mode of administration, andthe toxicity profile of the selected agent.

The ratio between toxicity and therapeutic effect for a particularcompound is its therapeutic index and can be expressed as the ratiobetween LD₅₀ (the amount of compound lethal in 50% of the population)and ED₅₀ (the amount of compound effective in 50% of the population).Therapeutic agents that exhibit high therapeutic indices are preferred.Therapeutic index data obtained from cell culture assays and/or animalstudies can be used in formulating a range of dosages for use in humans.The dosage of such compounds preferably lies within a range of plasmaconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. See, e.g. Fingl etal., In: THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, Ch. 1, p. 1, 1975.The exact formulation, route of administration, and dosage can be chosenby the individual physician in view of the patient's condition and theparticular method in which the therapeutic agent is used.

When parenteral application is needed or desired, particularly suitableadmixtures for the recombinant proteins described herein included in thepharmaceutical composition may be injectable, sterile solutions, oily oraqueous solutions, as well as suspensions, emulsions, or implants,including suppositories. In particular, carriers for parenteraladministration include aqueous solutions of dextrose, saline, purewater, ethanol, glycerol, propylene glycol, peanut oil, sesame oil,polyoxyethylene-block polymers, and the like. Ampoules are convenientunit dosages. Pharmaceutical admixtures suitable for use in thepharmaceutical compositions presented herein may include thosedescribed, for example, in Pharmaceutical Sciences (17th Ed., Mack Pub.Co., Easton, Pa.) and WO 96/05309, the teachings of both of which arehereby incorporated by reference.

Thus, in one aspect is a pharmaceutical composition that includes apharmaceutically acceptable excipient, a recombinant masking protein anda recombinant ligand protein. The recombinant masking protein includestwo identical masking protein domains. Each masking protein domainincludes: (1) a masking dimerizing domain as described herein, includingembodiments thereof; (2) a ligand-masking binding domain as describedherein, including embodiments thereof and (3) a cleavable masking linkeras described herein, including embodiments thereof, that connects theligand-masking binding domain to the masking dimerizing domain. Themasking protein domains are bound together. The recombinant ligandprotein includes two identical ligand protein domains as describedherein, including embodiments thereof. Each ligand protein domainincludes: (1) a ligand dimerizing domain as described herein, includingembodiments thereof; (2) a ligand domain as described herein, includingembodiments thereof; and (3) a ligand linker connecting the liganddomain to the ligand dimerizing domain as described herein, includingembodiments thereof. The ligand protein domains are bound together. Inembodiments, the ligand domain is bound to one of said ligand-maskingdomains.

In embodiments, the masking dimerizing domains of the pharmaceuticalcomposition are connected to a targeting domain through a targetinglinker. The targeting domain and targeting linker are as describedhereinabove in the Compositions section. In embodiments, the targetinglinker is a cleavable targeting linker as described hereinabove in theCompositions section.

In embodiments, the ligand dimerizing domains of the pharmaceuticalcomposition are connected to a targeting domain through a targetinglinker. The targeting domain and targeting linker are as describedhereinabove in the Compositions section. In embodiments, the targetinglinker is a cleavable targeting linker as described hereinabove in theCompositions section.

The pharmaceutical composition of the compositions described herein maybe supplied in an administration device, such as a syringe, pen, or jetinjector. In embodiments, the pharmaceutical compositions describedherein may be supplied as an aqueous suspension or as a powder within avessel (which may optionally be reconstituted with a suitable diluentsuch as buffers, saline, or water). The vessel may be a storage device,such as an intravenous bag or other readily usable container capable ofstoring and protecting the pharmaceutical compositions containedtherein. The vessel may be an administration device, such as a syringe,pen, or jet injector. When the vessel is a syringe or a storage devicesuch as an intravenous bag, the pharmaceutical compositions describedherein may be supplied in a concentration or dose ready for use. Thepharmaceutical compositions described herein may be supplied as a kit asdescribed herein.

III. KITS

Also provided herein are kits. In one aspect, the kits herein include arecombinant masking protein and a recombinant ligand protein. Therecombinant masking protein includes two identical masking proteindomains. Each masking protein domain includes: (1) a masking dimerizingdomain as described herein, including embodiments thereof; (2) aligand-masking binding domain as described herein, including embodimentsthereof; and (3) a cleavable masking linker as described herein,including embodiments thereof, that connects the ligand-masking bindingdomain to the masking dimerizing domain. The masking protein domains arebound together. The recombinant ligand protein includes two identicalligand protein domains as described herein, including embodimentsthereof. Each ligand protein domain includes: (1) a ligand dimerizingdomain as described herein, including embodiments thereof; (2) a liganddomain as described herein, including embodiments thereof; and (3) aligand linker connecting the ligand domain to the ligand dimerizingdomain as described herein, including embodiments thereof. The ligandprotein domains are bound together. In embodiments, the ligand domain isbound to one of said ligand-masking domains.

The recombinant masking protein and the recombinant ligand protein,including components thereof, are as described hereinabove the incompositions section. The recombinant masking protein and recombinantligand protein may be in one container. The recombinant masking proteinand recombinant ligand protein may be in separate containers. Whenprovided in separate containers, the recombinant masking protein and therecombinant ligand protein may be mixed together and administered usingthe methods provided herein. The ligand domain of the recombinant ligandprotein may be bound to one of the ligand masking domains of therecombinant masking protein.

The composition of the kit may be supplied in an administration device,such as a syringe, pen, or jet injector. The compositions of the kit maybe supplied as an aqueous suspension or as a powder within a vessel(which may optionally be reconstituted with a suitable diluent such asbuffers, saline, or water). The vessel may be a storage device, such asan intravenous bag or other readily usable container capable of storingand protecting the compositions contained therein. The vessel may be anadministration device, such as a syringe, pen, or jet injector. When thevessel is a syringe or a storage device such as an intravenous bag, thepharmaceutical compositions described herein may be supplied in aconcentration or dose ready for use. The pharmaceutical compositionsdescribed herein may be supplied as a kit as described herein.

IV. METHODS

Also provided herein are methods of treating a disease in a subject inneed thereof. In one aspect, the method includes administering to asubject in need thereof a therapeutically effective amount of arecombinant masking protein as described herein, including embodimentsthereof and a recombinant ligand protein as described herein, includingembodiments thereof. In embodiments, the recombinant masking protein andthe recombinant ligand protein are administered simultaneously to thesubject in need. In embodiments, the recombinant masking protein and therecombinant ligand protein are administered sequentially to the subjectin need (e.g. within about 1, 2, 3, 4, 5, 6 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 1, 1, 19, 20, 25, 30, 35, 40, 45, 50, 55, or 60 min of eachother). In embodiments, the recombinant masking protein and therecombinant ligand protein are administered within about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 28, 32,36, 40, or 48 hours of each other.

In embodiments, the recombinant masking protein and the recombinantligand protein are combined and/or administered as a pharmaceuticalcomposition described herein, including embodiments thereof. Inembodiments, the recombinant protein and recombinant ligand protein aresupplied in a kit before administration to the subject in need thereof.

In embodiments of the methods herein, the ligand domain may be bound toat least one of the ligand-masking domains prior to administration. Therecombinant masking protein and/or the recombinant ligand protein mayindependently include a targeting domain connected to the maskingdimerizing domain or ligand dimerizing domain respectively through atargeting linker. The targeting domain and targeting linker are asdescribed herein, including embodiments thereof. In embodiments, thetargeting linker is a cleavable targeting linker as described herein,including embodiments thereof (e.g. the cleavable targeting linkerincludes a protease cleavage site).

In embodiments, the methods herein provide for decreased adverse effectsassociated with monoclonal antibody therapies. Thus, administration ofthe compositions herein may reduce acneiform eruptions, enterocolitis,dermatitis, hypophysitis, uveitis, hepatitis, nephritis,gastrointestinal irritation, cardiotoxicity, or development ofprogressive multifocal leukoencephalopathy. In embodiments, the methodsinclude a reduction of co-administration of an immunosuppressant (e.g.immunosuppressant administered with monoclonal antibody therapies tolimit autoimmune disorders). Thus, the compositions herein may beadministered alone or be coadministered to the patient. Coadministrationis meant to include simultaneous or sequential administration of thecompound individually or in combination (more than one compound oragent). Thus, the preparations can also be combined, when desired, withother active substances (e.g. anticancer agents).

The compositions of the present invention can be deliveredtransdermally, by a topical route, formulated as applicator sticks,solutions, suspensions, emulsions, gels, creams, ointments, pastes,jellies, paints, powders, and aerosols. Oral preparations includetablets, pills, powder, dragees, capsules, liquids, lozenges, cachets,gels, syrups, slurries, suspensions, etc., suitable for ingestion by thepatient. Solid form preparations include powders, tablets, pills,capsules, cachets, suppositories, and dispersible granules. Liquid formpreparations include solutions, suspensions, and emulsions, for example,water or water/propylene glycol solutions. The compositions of thepresent invention may additionally include components to providesustained release and/or comfort. Such components include high molecularweight, anionic mucomimetic polymers, gelling polysaccharides andfinely-divided drug carrier substrates. These components are discussedin greater detail in U.S. Pat. Nos. 4,911,920; 5,403,841; 5,212,162; and4,861,760. The entire contents of these patents are incorporated hereinby reference in their entirety for all purposes. The compositions of thepresent invention can also be delivered as microspheres for slow releasein the body. For example, microspheres can be administered viaintradermal injection of drug-containing microspheres, which slowlyrelease subcutaneously (see Rao, J. Biomater Sci. Polym. Ed. 7:623-645,1995; as biodegradable and injectable gel formulations (see, e.g., GaoPharm. Res. 12:857-863, 1995); or, as microspheres for oraladministration (see, e.g., Eyles, J. Pharm. Pharmacol. 49:669-674,1997). In embodiments, the formulations of the compositions of thepresent invention can be delivered by the use of liposomes which fusewith the cellular membrane or are endocytosed, i.e., by employingreceptor ligands attached to the liposome, that bind to surface membraneprotein receptors of the cell resulting in endocytosis. By usingliposomes, particularly where the liposome surface carries receptorligands specific for target cells, or are otherwise preferentiallydirected to a specific organ, one can focus the delivery of thecompositions of the present invention into the target cells in vivo.(See, e.g., Al-Muhammed, J. Microencapsul. 13:293-306, 1996; Chonn,Curr. Opin. Biotechnol. 6:698-708, 1995; Ostro, Am. J. Hosp. Pharm.46:1576-1587, 1989). The compositions of the present invention can alsobe delivered as nanoparticles.

The method may include allowing a portion of the recombinant maskingprotein and the recombinant ligand protein to localize to a cancer cellor tumor. The may include allowing a cleaving agent (e.g. a protease) tocleave one or more of the cleavage sites (e.g. the cleavage site of thecleavable masking linker, the cleavable ligand linker, or cleavabletargeting linker).

In embodiments, the disease is cancer, an autoimmune disease, aneurodegenerative disease, or a cardiovascular disease. The cancer maybe leukemia, lymphoma, head and neck cancer, colorectal cancer, prostatecancer, melanoma, breast cancer or neuroblastoma. The cancer may beleukemia. The cancer may be lymphoma. The cancer may be head and neckcancer. The cancer may be colorectal cancer. The cancer may be prostatecancer. The cancer may be melanoma. The cancer may be breast cancer. Thecancer may be neuroblastoma. The recombinant masking protein andrecombinant ligand protein may be co-administered with other anti-canceragents described herein.

In certain embodiments, the cancer cell forms part of a tumor, whereinthe cleaving agent (e.g. a protease) may be located proximal to thecancer cell. The linkers described herein may be cleaved at the cleavagesite. Thus, if the linker is a cleavable masking linker, the linker iscleaved at the cleavage site and the ligand-masking domain is freed. Ifthe linker is a targeting linker, the linker may be cleaved at thecleavage site and the targeting domain is freed. The cleaving agent maybe a protease. The cleaving agent may be matrix metalloproteinase (MMP).The MMP may be MMP-2 or MMP-9.

In embodiments, the autoimmune disease is Crohn's disease, rheumatoidarthritis, asthma, or psoriasis. The autoimmune disease may be Crohn'sdisease. The autoimmune disease may be rheumatoid arthritis. Theautoimmune disease may be asthma. The autoimmune disease may bepsoriasis.

The neurodegenerative disease may be Alzheimer's disease or multiplesclerosis. The neurodegenerative disease may be Alzheimer's disease. Theneurodegenerative disease may be multiple sclerosis.

V. SEQUENCES

CTLA4-Fc_WT: (SEQ ID NO: 1)MHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDGSRSGGTSGGGSVPLSLYSGSTSGSGKSSEGSGQASTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. E31A_R33A:(SEQ ID NO: 2)MHVAQPAVVLASSRGIASFVCEYASPGKATAVAVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDGSRSGGTSGGGSVPLSLYSGSTSGSGKSSEGSGQASTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >E31S: (SEQ ID NO: 3)MHVAQPAVVLASSRGIASFVCEYASPGKATSVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDGSRSGGTSGGGSVPLSLYSGSTSGSGKSSEGSGQASTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. E31K: (SEQ ID NO: 4)MHVAQPAVVLASSRGIASFVCEYASPGKATKVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDGSRSGGTSGGGSVPLSLYSGSTSGSGKSSEGSGQASTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. E31R: (SEQ ID NO: 5)MHVAQPAVVLASSRGIASFVCEYASPGKATRVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDGSRSGGTSGGGSVPLSLYSGSTSGSGKSSEGSGQASTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. E31S_R33E_T51H:(SEQ ID NO: 6)MHVAQPAVVLASSRGIASFVCEYASPGKATSVEVTVLRQADSQVTEVCAAHYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDGSRSGGTSGGGSVPLSLYSGSTSGSGKSSEGSGQASTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. T51H: (SEQ ID NO: 7)MHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAAHYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDGSRSGGTSGGGSVPLSLYSGSTSGSGKSSEGSGQASTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. K95A: (SEQ ID NO: 8)MHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICAVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDGSRSGGTSGGGSVPLSLYSGSTSGSGKSSEGSGQASTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. E97A: (SEQ ID NO: 9)HVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVALMYPPPYYLGIGNGTQIYVIDPEPCPDSDGSRSGGTSGGGSVPLSLYSGSTSGSGKSSEGSGQASTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. K95A_E97A:(SEQ ID NO: 10)MHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICAVALMYPPPYYLGIGNGTQIYVIDPEPCPDSDGSRSGGTSGGGSVPLSLYSGSTSGSGKSSEGSGQASTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

VI. EXAMPLES 1. Example 1

Despite major advances, locally advanced and metastatic prostate cancerremain a clinical challenge. In contrast to early, non-disseminatedprostate cancer, advance prostate cancer cannot be cured by currenttreatment modalities. In addition to chemotherapy, radiation and hormonetherapy, immunotherapy has recently left its mark on prostate cancermanagement (see the approval of Sipuleucel-T for the treatment oflate-stage prostate cancer). In other cancers, such as advancedmelanoma, dramatic improvements in overall survival of patients werereported with a monoclonal antibody (MDX-010, ipilimumab) thatrecognizes and blocks the immunosuppressive cytotoxic T-lymphocyteantigen (CTLA)-4 receptor on T cells. Hodi, 2010. These resultsultimately led to FDA-approval of ipilimumab for the treatment ofadvanced melanoma. Furthermore and of particular importance to thisapplication, phase-III clinical studies are currently being conducted toexplore the use of ipilimumab in advanced prostate cancer and otherneoplastic diseases.

However, ipilimumab can cause severe (grade 3-4) systemic adverse eventsreflecting immune-mediated toxicities. Beck, 2006; Phan, 2008; Weber,2008. These include enterocolitis, dermatitis, hypophysitis, uveitis,hepatitis, and nephritis. Enterocolitis is the most common majortoxicity (21% of patients) requiring extensive countermeasures includingcolectomy due to perforation of the colon in several patients. Beck,2006. The apparent risks of systemic CTLA-4 blockade raise questionsregarding its use. Herein, it was discovered, inter alia, that directingthese agents to tumor sites spares normal tissues and appears to be analternative and safer approach. This discovery enhances the therapeuticratio of CTLA-4 antagonists including ipilimumab and an engineeredlipocalin2 derivative recently developed as an effective CTLA-4antagonist. Schonfeld, 2009.

Most protein-based therapies currently in clinical use target molecularmechanisms but not disease sites. With few exceptions (such aslipocalin2) these are monoclonal antibodies (mAbs) which bind todiseased cells or to T cells (as in the case of ipilimumab). However,these monoclonal antibodies engage these targets in normal tissues andgive rise to adverse events with various degrees of severity. If thetherapeutic agent induces autoimmune phenomena, the toxicity not onlyleads to significant morbidity but often necessitates the administrationof immunosuppressants (corticosteroids, TNF-α inhibitors) which mayblunt therapeutic intent.

Thus, herein, the ‘targeted release’ of proteins at disease sites wasinvestigated. This concept relates to the reversible occlusion or‘reversible masking’ of reactive sites by ligands which can be removedby proteases present and active at disease sites. In the case of mAbs,the occluding moieties are recombinant antigen (Ag) fragments covalentlylinked to heavy or light IgG chains. In this design, the Ag fragmentoccludes the antigen binding sites on the IgG and, thus, preventsbinding to native antigen on target cells. However, the linker tetheringthe Ag fragment to the mAb contains protease cleavage sites which aresusceptible to tumor-associated proteases (for example matrixmetalloproteinases (MMPs)). Cleavage of the linker in the tumormicroenvironment reduces the strength (valency) of the intermolecularinteraction, dissociation of the complex and engagement of native Ag ontumor or resident normal cells. It has been previously demonstrated thatreversible masking of the EGFR antagonistic antibodies cetuximab (C225)and matuzumab (425) is feasible. Donaldson, 2009. However, these priorstudies were not directed to non-IgG molecules such as CTLA-4antagonists to restrict its immunostimulatory effects to the tumorenvironment (see FIG. 1A).

The CTLA-4 antagonist of choice is modified lipocalin2 (m-Lcn2) whichhas been optimized to engage CTLA and to reverse CTLA-4-dependentimmunosuppression. Schonfeld, 2009. M-Lcn2 was selected due to severalfavorable molecular characteristics. First, it is a stable solubleCTLA-4 ligand of which several variants with different affinities forCTLA-4 ranging from ˜100 nM to 250 pM have been identified. Secondly,the m-Lcn2 monomer is small (˜180 aa) as compared to IgG, yet it acts ina manner similar to ipilimumab as regards CTLA-4 antagonism. Thirdly,m-Lcn2 binds to and blocks both human and murine CTLA-4 enabling studiesin syngeneic mouse tumor models as proposed here. Lastly, detailedstructural knowledge of the m-Lcn2 interaction with CTLA-4 (Schonfeld,2009) allows design of a m-Lcn2/CTLA fragments that will readilydissociate upon MMP9 cleavage. Local CTLA-4 blockade at tumor sites haspreviously been tested by using B16 melanoma cells engineered to secretea CTLA-4 antagonistic antibody Simmons, 2008. The previous work showedthat local delivery of anti-CTLA-4 IgG secreted by GM-CSF-producingtumor cell-based vaccines activated potent anti-tumor responsesassociated with low circulating mAb levels in the host. Importantly,lowering the systemic exposure of the host to the anti-CTLA-4 alsocorrelated with reduced systemic autoimmunity.

Herein, are described different reversible recombinant masked m-Lcn2variants and testing of their immunostimulatory properties in vitro andin vivo using appropriate mouse cancer models.

Different versions of CTLA-4 antagonists tested provide effective CTLA-4blockade at tumor sites while exhibiting a reduced adverse side effectprofile compared to systemically active CTLA-4 antagonists. m-Lcn2, are-engineered human lipocalin that binds both murine and human CTLA4with high affinity and which enables us to translate successful outcomesin murine studies to human clinical trials will be the focus. m-Lcn2 ismonovalent whereas ipilumumab (and nearly all approved mAb-basedtherapeutics) are bivalent. Thus, to mimic ipilumumab and leverageaffinity gains by avidity, a bivalent analog was created (FIG. 2A).Specifically, we fused m-Lcn2 to a human IgG1 Fc-fragment.

The bivalent nature of m-Lcn2-Fc is also critical in creating anon-covalent tumor-activated, bivalent mask. To create the m-Lcn2 mask,CTLA-4 was fused to the N-terminus of the IgG1 Fc through a flexiblelinker encoding a protease recognition sequence (MMP9) that is cleavedby a protease (MMP9) present and active at prostate cancer sites (FIG.2B). Cleavage of the MMP9 linker reverts the valency of the mask to amonomeric interaction, significantly reduces the affinity of the maskingmoiety, and permits the bivalent m-Lcn2 to associate with TIL-associatedCTLA-4 (FIG. 1). The cleaved, monomeric CTLA-4, which cannot competewith endogenous, multimeric CTLA-4, is predicted to diffuse away fromthe tumor microenvironment (see FIG. 1).

By reversibly masking m-Lcn2, systemic administration to disease sitesis achieved by cleavage of the mask and differential affinity at thetumor site. To add a ‘second layer’ of tumor targeting, we fused scFvrecognizing a tumor-specific antigen to either the m-Lcn2 or to the mask(FIG. 2C/D). Without being bound by any particular theory, wehypothesize that anchoring the masked m-Lcn2 complex at the tumor sitewill increase the residence time at the tumor, enhance its proteolyticcleavage and facilitate unmasking. To accomplish this, scFv will befused to the C-terminus of the Fc (Czajkowsky, 2012) through a Gly-Serlinker with and without an MMP9 site. To enable in vivo studies in anappropriate prostate cancer mouse model (ProTRAMP mice), we will use ahemagglutinin (HA) scFv. Zhang, 2010.

Separating the mask from the m-Lcn2-Fc simplifies the optimization andcharacterization of the therapeutic moiety (e.g., m-Lcn2-Fc). The linkerand the protease site, can be independently matched and optimized to thetumor. Using a Fc domain for dimerization is advantageous, in part dueto the fact that Fc containing biologic agents (mAbs) constructs are inclinical use, but also since Fc fusions frequently express well, areeasily purified using protein A/L resins, and offer favorable PK/PDproperties.

Collectively, these constructs described here provide a broad platformto test whether antagonizing CTLA-4 expressed by TILs at tumor sites isfeasible and effective in mitigating adverse side effects associatedwith this treatment modality.

Before creating the bivalent m-Lcn2-Fc construct, we first verified thatthe monovalent m-Lcn2 binds to human CTLA4 as described by Skerra andcolleagues. Schonfeld, 2009. The m-Lcn2 cDNA was commerciallysynthesized and subcloned into a bacterial expression vector. Theprotein was isolated from the periplasm and purified to homogeneity bystandard methods (>95% by SDS-PAGE). The binding affinity of thisconstruct was determined using surface plasmon resonance (SPR) whereinCTLA-4-Fc (purchased from R&D systems) was chemically tethered to CMSchip. The affinity was essentially identical to the reported value.Next, the same m-Lcn2 cDNA was subcloned N-terminal to a linker-Fedomain present in an insect expression vector. Viral stocks wereproduced, titrated and used to express the construct. The secretedproduct was purified to homogeneity using standard methods. The sameprocess was carried out for the CTLA-4 mask. The MMP9 site wasincorporated through site directed mutagenesis.

To verify that the m-Lcn2-Fc and CTLA-4-Fc form a 1:1 complex, we usedanalytical size exclusion chromatography (SEC) (FIG. 3A). A definitiveshift to earlier elution volumes was observed, consistent with amolecular mass of 200 kD, as expected. To verify an increase in theapparent affinity due to the energy additivity of bivalent-bivalentinteraction, the mask, CTLA-4-MMP9-Fc, was coupled to a CMS sensor chipand titrated with the bivalent m-Lcn2-Fc. A substantial increase inaffinity was observed. In fact, no dissociation was observed and thus nocalculation could be completed for an apparent dissociation constant(FIG. 3B). An approximate upper limit is estimated at K_(D)=200 pM. Inan attempt to establish the off-rate, a competition assay was carriedout. Specifically, the 1:1 m-Len2-Fc|CTLA-4-MMP9-Fc complex was isolatedand incubated with 10 fold excess of Alexafluor-labeled CTLA-4-MMP9-Fc.The exchange by size exclusion chromatography was followed by monitoringat 495 nM. After two weeks, less than 5% exchange was observed. Takentogether, these results suggest that the bivalent m-Lcn2-Fc/CTLA-4-Fccomplex is stable well beyond its predicted biological half-life (<3-5days) upon administration in vivo.

Recombinant MMP9 was demonstrated to effectively cleave theCTLA4-MMP9-Fc mask (FIG. 3C), but not the m-Lcn2-Fc. To enhance thedissociation rate after MMP9 cleavage, we superimposed the structures ofCTLA-4 bound to CD80, CD86 and m-Lcn2. Based on this superposition,mutations in CTLA-4 were identified that can be introduced individually(or in combination to lower the affinity of the m-Lcn2 mask andsimultaneously reduce the affinity of the cleaved CTLA-4 tocell-associated CD80/86 (FIG. 4). These include Thr32, Glu33, Tyr100 andTyr104. Each is at the Lcn2 and CD80/86 interface and each is surfaceexposed.

Each will be mutated to alanine, expressed, purified and characterizedby SPR (monovalent m-Lcn2 and m-Lcn2-Fc will be chemically tethered toindividual channels of the SPR chip). In particular, we seek to reducethe lifetime of the monovalent interaction (K_(OFF)=5.1×10⁻⁴s⁻¹;t_(1/2)=0.693/K_(OFF)=22 mins). A 10-fold increase in the off-rate wouldmitigate potential loss of the tumor localization of the m-Lcn2-Fc dueto diffusion back to the blood stream. Reducing the affinity of theindividual interactions will affect the overall affinity of the bivalentmask to the bivalent m-Lcn2-Fc (e.g., 100 fold weaker). Due tosignificant gains in affinity due to avidity, we expect efficientmasking (e.g., 320 nM×320 nM=100 fM), however, we will immediatelyobserve significant losses in the SPR experiments. Such losses reflectpotential issues within the linker (e.g., too long, too flexible, toorestricted). Issues with the linkers require creating a series oflinkers between the Fc and the CTLA4 portions of the mask. First, wereduce the length in an iterative fashion (e.g., remove 2 residues percycle until we lose avidity). Next, we alter the amino acid sequence.The linker is composed primarily of glycines beyond the MMP9 cleavagesite. To reduce the flexibility inherent in glycine repeat sequences, weadd serines, threonines and/or glutamines. In each iteration, we testthe affinity by SPR and the MMP9 susceptibility of the complex.

The cleavage of the mask by MMP9 will be optimized. Given the apparentaffinity of the bivalent mask is sufficient, we alter the position ofthe MMP9 site. Specifically, we move the site to the N-terminal andC-terminal position of the linker. We compare the proteolytic rate ofeach in the presence of the m-Lcn2-Fc. The linker may be extended tomake the site more solvent exposed. We add residues to the N- andC-termini of the linker. As before, each will be characterized by SPR toensure efficient masking.

An scFv that recognizes HA will be fused to the C-terminus of the Fcdomain in both the m-Lcn2-Fc construct and the CTLA-4 mask. Zhang, 2010.The inclusion of this domain allows tumor targeting, but alsosufficiently extends the residence time at the tumor for efficientproteolysis. It also counteracts extended off-rates of the mask. ThecDNA will be commercially synthesized and subcloned into the currentconstructs. We characterize by SPR the affinity the scFv to HA andensure that its presence does not affect the binding of Lcn2 for CTLA-4and vice versa. We will also characterize MMP9 proteolytic cleavage.Similar iterations as outlined above will be carried out if necessary.

We test functionally whether masking of m-Lcn2 disrupts its ability toboost T cell activation and whether unmasking restores T cellactivation. This can be done using in vitro assays of T cell activation.In vitro screening is a cost-effective way to select optimizedcandidates for in vivo testing of anti-tumor effects and immuneactivation. Specifically we test whether the m-Lcn2 construct enhancesnaive and antigen-specific (OT-1) T cell activity. In addition to maskedconstructs we test constructs targeted to prostate cells by linkingscFvs recognizing hemagglutinin expressed selectively in the prostate.We assess the effect of masking/tumor cell targeting of m-Lcn2 variantson immune effector functions in the presence and absence of recombinantMMP9 as appropriate and by using controls in which linkers do notcontain MMP-9 cleavable sites.

Previous work has shown that CTLA-4 antagonistic antibodies augmentefficacy of immunotherapeutic approaches to mouse tumors including B16melanoma (Quezada, 2006; Curran, 2010; Korman, 2005; Gregor, 2004) andTRAMP-C2 prostate cancer cells (Kwon, 1999; Waitz, 2012). SpecificallyWaitz, et al showed effective but not complete growth inhibition ofsubcutaneous TRAMP-C2 grafts in C57/B16 mice by a combination ofcryoablation and antibody-mediated CTLA-4 blockade. By contrast, CTLA-4blockade alone was insufficient for tumor growth inhibition. Based onthese findings, the TRAMP-C2 implant model is highly suitable forassaying anti-tumor effects of cleavable and uncleavable m-Lcn2constructs alone or in combination with cryoablation. This model willlikely not accommodate testing the masked m-Lcn2-Fc design equipped withtumor-targeted sc-Fvs as we do not have access to antibodies recognizingtumor-associated antigens on TRAMP cells and SV40T is not detectable onthe cell surface of TRAMP prostate epithelial cells.

Instead, we use a different model system generated to avoid theseissues. TRAMP mice were crossed with mice expressing hemagglutinin undercontrol of the probasin promoter. See Getnet, 2009; Grosso, 2007. Inthis double transgenic model (referred to in the following as ProTRAMP),hemagglutinin represents a prostate-associated antigen co-expressed withSV40T in both normal and transformed parenchymal prostate cells.Therefore, tumor tissue targeting will be achieved by C-terminallylinking HA-specific scFvs onto the Fc fragment of the mLcn-2Fcconstruct.

In addition to testing efficacy of masked, cleavable m-Lcn2 constructswe focus on (i) monitoring potential adverse events induced by CTLA-4blockade in mice (ii) testing whether masking m-Lcn2 reduces suchadverse effects and, (iii) assessing redistribution of m-Lcn2 to tumortissues. Regardless of the extent of adverse events associated withm-Lcn2 administration, this model system provides important informationon biodistribution of masked m-Lcn2 constructs critical to the avoidanceor reduction of adverse events in patients. Tumor-specific immuneresponses in all experimental and control animals will be assessed andas shown in Table 1. Novak, 2007.

To assess how m-Lcn2-mediated blockade of cell-associated CTLA-4 affectsantigen-specific activation of the T cells, we will generate TRAMP-C2cells expressing chicken ovalbumin (Ova) reporter by stable transductionof the cells with a plasmid encoding full-length Ova. The resultantTRAMP-C2-Ova cells will be used for the analysis of the IFNγ productionand cytotoxic T lymphocyte (CTL) activity of Ova-SIINFEKLpeptide-specific T cells isolated from OT-I transgenic mice. Productionof IFNγ by the activated T cells in the absence or presence of theCTLA-4 blockade will be assessed by ELISpot assay a commercial kit(eBioscience). Analysis of CTL activity will be done using TRAMP-C2-Ovaactivated OT-I cells as described below for B16 mouse melanoma cells(Table 2). All assays will be done in triplicates using OT-I cellsactivated in vitro by the pre-incubation with TRAMP-C2-Ova cells in theabsence of presence of the recombinant proteins under investigation.

TABLE 2 T-cell activation by masked and unmasked m-Lcn2-Fc proteins. Invitro stimulation assay B16-OVA 15 14 B16-OVA + m-Lcn2-Fc 167 159B16-OVA + masked m-Lcn2-Fc 24 20 *Elispot assays (IFN-γ) were performedfollowing MLR using splenocytes isolated from OT-1 (ovalbumin-specificTCR transgenic) mice after stimulation in vitro (5 d) with B16 melanomacells expressing Ova peptide in the presence and absence of recombinantmasked and unmasked m-Lcn2 constructs as indicated and as shown inFIG. 1. Note 10-fold induction of IFN-γ production by unmasked but notby masked m-Lcn2-Fc.

We have tested the effects of masked and unmasked prototypic m-Lcn2-Fcconstructs (FIG. 1) using Ova-expressing B16 melanoma cells andsplenocytes derived from OT-1 mice (Table 2). This experiment revealed amarked increase in IFNγ production associated with bivalent m-Lcn2-Fctreatment which was reversible to almost background levels by use of thebivalent CTLA-4 mask. Similar results were obtained using splenocytesfrom naive mice stimulated with phetohemagglutinin. Based on these verypromising results we anticipate to advance at least two masked m-Lcn2designs in mice. One of these is a version of the prototypical designshown in FIG. 1A, whereas the second will be one of the tumor-targeteddesign containing anti-HA scFvs and schematically shown in FIG. 2.Selection of the optimized construct will be done by a combination ofbiophysical (SPR) and functional assays.

Effects of Reversibly Masked m-Lcn2 on TRAMP-C2 Growth in C56BL/6 Mice.

To assess efficacy of the non-targeted, masked m-Lcn2-Fc/CTLA4-Fccomplexes we will use a model based on inhibition of TRAMP-C2 cells uponsubcutaneous grafting to male C57BL/6 mice. Waitz, 2012. Mycoplasma-free(PCR-tested) TRAMP-C2 cells (1×10⁶) will be inoculated subcutaneouslyinto the left flank of mice. After 28 d tumors will be cryoablatedfollowed by a 2^(nd) inoculation of 0.2×10⁶ TRAMP-C2 cells in the rightflank. Masked and unmasked m-Lcn2-Fc proteins will be injected on days1, 4, 7, and 10 after the 2^(nd) inoculation and tumor growth monitoredfor further 60 days. Controls will consist of constructs and complexesrendered MMP9 resistant by introducing point mutations in the MMPconsensus site; these variants proteins are not expected to inhibittumor growth. Hamster IgG (negative control) and the anti-mouse 9H10antibody (positive control) are purchased from BD Biosciences. We willuse 100-200 μg of IgG/injection and equimolar concentrations of maskedand unmasked recombinant m-Lcn2-Fc proteins.

In this model neither single modality cryoablation nor anti-CTLA-4treatment protect and all mice die within 30 days of the 2^(nd) tumorcell inoculation. Waitz, 2012. In contrast, cryoablation combined withCTLA-4 blockade by 9H10 confers long-term protection (>60 days) toapproximately 50% of mice. We expect the cleavable masked and unmaskedm-Lcn2 constructs produce long-term protection. By contrast theuncleavable versions of these proteins should not provide anyprotection. If protection is observed in this setting we plan to alsotest whether protection can be achieved by recombinant, cleavable m-Lcn2alone in the absence of cryoablation. As TRAMP-C2 cells overexpress MMP9we will perform these assays with unmodified TRAMP-C2s. If necessary wewill transfect TRAMP-C2 cells with a plasmid encoding MMP9 and use cellssecreting high levels of active MMP9.

Independently of the potential therapeutic effect of masked m-Lcn2 wewill determine whether reversible masking leads to reduced incidence orseverity of autoimmune parameters in mice treated with CTLA-4antagonists. Quantification of “autoimmune” status in treated mice willbe done by determining the levels of autoantibodies reactive withanti-nuclear antibodies ANA, sSDNA, and dsDNA using commercial ELISAkits. These parameters were chosen based on previous work assessingsystemic immune related events associated with vaccination strategies inmice (Hodge, 2003) and showing significant increases of these antibodiesin the circulation when CTLA-4 antagonistic antibody was systemicallyadministered to mice (Simmons, 2008). These experiments will revealwhether ‘naked’ m-Lcn2 triggers systemic immune responses in a mannersimilar to CTLA-4 antagonistic antibodies and whether reversibly maskedm-Lcn2 will reduce systemic immune responses. Serum will be collected atseveral time points within 2 weeks of m-Lcn2 variant proteinadministration. We will also assay biodistribution of cleavable andnoncleavable m-Lcn2 in TRAMP-C2 tumor tissue in mice to documentinteraction of unmasked m-Lcn2 interacts with TILs in situ. This will bedone by double staining tissue sections for T cell surface markers (CD4,CD8) and m-Lcn2 using an antibody recognizing the His-tag which is notremoved by MMP9. We will further detect cleaved and uncleaved m-Lcn2 insera of treated mice by immunoblot analysis.

Second generation unmasked or masked m-Lcn2 constructs targeted toprostate cancer will be tested in vivo using ProTRAMP mice in whichprostate epithelia express the model influenza hemagglutinin undercontrol of a prostate-specific minimal rat probasin promoter (Drake,2005 #1818). Using this model will make it possible to monitor immunephenomena associated with CTLA-4 blockade directed to tumor/tissue sitesas it leads to intraprostatic infiltration and accumulation ofclonotypic, HA-specific cytotoxic T-cells. Drake, 2005. This model alsomimics the situation of several prostate cancer-associated antigens inhuman prostate cancer which would ultimately serve to target recombinantCTLA-4 antagonists to human disease sites.

We will use this model to demonstrate enrichment of anti-HA-enabledCTLA-4 antagonists in the prostate. This will be done by i.p. injectionof these and control recombinant proteins that do not contain anti-HAscFv sequences into tumor-bearing animals (>10-weeks old) followed bydetection of these in different tissues using commercially availableanti-His and anti-human Fc antibodies.

Secondly, we will test whether targeting m-Lcn2-Fc or monomeric m-Lcn2to the prostate delays or inhibits tumor development in ProTRAMP mice.These mice develop tumors with similar kinetics as TRAMP mice. Drake,2005. To assay effects of CTLA-4 blockade on tumor development in thesemice we plan to take advantage of a mouse model to monitor abscopaleffects of radiation therapy in combination with immunotherapy. Harris,2008. Specifically we will perform pelvic irradiation of 20-24 week oldProTRAMP mice followed by CTLA-4 antagonist administration within 3 to 5weeks after irradiation. This time regimen has been found by Harris etal. to be superior to others in enabling anti-tumor immune responses inProTRAMP mice.

It is expected that the scFv-enabled m-Lcn2 proteins (monomers an orFc-enabled dimers) preferentially localize to the prostate and are moreeffective in inhibiting the development of spontaneous prostate cancerin this model.

The antigen binding site of trastuzumab, domain IV of the Her2 receptor,was tethered to the dimeric Fc region of the human IgG1 through aflexible peptide linker containing a tumor-specific protease site, MMP.This approach offered significant advantages over the covalent maskedantibodies, because the non-covalent Fc masks can be added to mAbsalready in the clinic. This mitigates the testing and optimization ofmodified mAbs. Further, a single cleavage of the non-covalent maskreduces the valency of the mask and initiates immediate release the mAb.

2. Example 2

Boxshade alignment CTLA4-Fc_WT 1

E31S 1

T51H 1

E31A_R33A 1

E31S_R33E_T51H 1

CTLA4-Fc_WT 61

E31S 61

T51H 61

E31A_R33A 61

E31S_R33E_T51H 61

CTLA4-Fc_WT 121

E31S 121

T51H 121

E31A_R33A 121

E31S_R33E_T51H 121

CTLA4-Fc_WT 181

E31S 181

T51H 181

E31A_R33A 181

E31S_R33E_T51H 181

CTLA4-Fc_WT 241

E31S 241

T51H 241

E31A_R33A 241

E31S_R33E_T51H 241

CTLA4-Fc_WT 301

E31S 301

T51H 301

E31A_R33A 301

E31S_R33E_T51H 301

CTLA4-Fc_WT 361

E31S 361

T51H 361

E31A_R33A 361

E31S_R33E_T51H 361

3. Example 3

CLUSTAL 2.1 multiple sequence alignment CTLA4-Fc_WTMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTF  60E31A_R33A MHVAQPAVVLASSRGIASFVCEYASPGKATAVAVTVLRQADSQVTEVCAATYMMGNELTF 60 E31S MHVAQPAVVLASSRGIASFVCEYASPGKATSVRVTVLRQADSQVTEVCAATYMMGNELTF 60 T51H MHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAAHYMMGNELTF 60 E31S_R33E_T51HMHVAQPAVVLASSRGIASFVCEYASPGKATSVEVTVLRQADSQVTEVCAAHYMMGNELTF  60****************************** * ***************** ********* CTLA4-Fc_WTLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPC 120E31A_R33A LDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPC120 E31S LDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPC120 T51H LDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPC120 E31S_R33E_T51HLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPC 120************************************************************ CTLA4-Fc_WTPDSDGSRSGGTSGGGSVPLSLYSGSTSGSGKSSEGSGQASTHTCPPCPAPELLGGPSVFL 180E31A_R33A PDSDGSRSGGTSGGGSVPLSLYSGSTSGSGKSSEGSGQASTHTCPPCPAPELLGGPSVFL180 E31S PDSDGSRSGGTSGGGSVPLSLYSGSTSGSGKSSEGSGQASTHTCPPCPAPELLGGPSVFL180 T51H PDSDGSRSGGTSGGGSVPLSLYSGSTSGSGKSSEGSGQASTHTCPPCPAPELLGGPSVFL180 E31S_R33E_T51HPDSDGSRSGGTSGGGSVPLSLYSGSTSGSGKSSEGSGQASTHTCPPCPAPELLGGPSVFL 180************************************************************ CTLA4-Fc_WTFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV 240E31A_R33A FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV240 E31S FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV240 T51H FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV240 E31S_R33E_T51HFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV 240************************************************************ CTLA4-Fc_WTVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ 300E31A_R33A VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ300 E31S VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ300 T51H VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ300 E31S_R33E_T51HVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ 300************************************************************ CTLA4-Fc_WTVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV 360E31A_R33A VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV360 E31S VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV360 T51H VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV360 E31S_R33E_T51HVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV 360************************************************************ CTLA4-Fc_WTFSCSVMHEALHNHYTQKSLSLSPGK- 385 E31A_R33A FSCSVMHEALHNHYTQKSLSLSPGK- 385E31S FSCSVMHEALHNHYTQKSLSLSPGK- 385 T51H FSCSVMHEALHNHYTQKSLSLSPGK- 385E31S_R33E_T51H FSCSVMHEALHNHYTQKSLSLSPGK- 385 *************************

4. Example 4

Wildtype trastuzumab mask No. 1. Human Her2 (underline); The Fc humanIgG1 (bold); Cleavage site (e.g MMP9 site) (bold underline):

(SEQ ID NO: 11) CSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQ PCPINGSRSGGTSGGGSVPLSLYS GSTSGSGKSSEGSGQASTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Wildtype trastuzumab mask No. 2. Human Her2 (underline); the Fc humanIgG1 (bold); Cleavage site (e.g MMP9 site) (bold underline):

(SEQ ID NO: 12) CSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQ PCPINGSRSGGTSGGGSVPGSGSS GSTSGSGKSSEGSGQASTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Mutated EGFR domain III heavy chain mask (N-terminus of the heavy chainof Cetuximab). The point mutations prevent “aggregation” (i.e., keepingthe mask from binding to cetuximab itself) EGRF domain III (underline);mutations (bold); cleavage site (MMP9 site) (bold underline):

(SEQ ID NO: 13) MRPSGTAGAALLALLAALCPASRARKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIAAWPENRTDLHAFENLEIIRGRTNMDGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPKDCVSCRNVSRGRECSRGGGSGGG SGGGS VPLSLYSGSTSGSGKSSEGSGSGAQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK.

Mutated EGFR Domain III Light Chain Mask:

(SEQ ID NO: 14) MRPSGTAGAALLALLAALCPASRADILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GA.

The Modified Lipocalin2 (mLcn2):

(SEQ ID NO: 15) Q D S T S D L I P A P P L S K V P LQ Q N F Q D N Q F H G K W Y V V G L A GN R I L R D D Q H P M N M Y A T I Y E LK E D K S Y N V T S V I S S H K K C E YT I A T F V P G S Q P G E F T L G N I KS Y G D K T S Y L V R V V S T D Y N Q YA V V F F K L A E D N A E F F A I T I YG R T K E L A S E L K E N F I R F S K SL G L P E N H I V F P V P I D Q C I D G

5. Example 5

The bivalent mask consists of two extracellular domains of human CTLA4fused via protease substrate to an IgG1 (FIG. 5). Briefly, the maskedmAb prodrug incorporated a non-covalent mask to occlude theantigen-binding region (CDR) of antibodies. In this design, thenon-covalent mask consisted of recombinant antigen fragments fused tohuman IgG1 through a flexible peptide linker containing tumor-associatedprotease substrate. The bivalent-bivalent avidity of the mAb:maskinteraction attains very high affinities. Expressed mathematically:

AG _(total) =AG ₁ +AG ₂ −AG _(linker)

where AG_(total) is the change in free energy of the total system, AG₁and AG₂ are the free energies of each CDR:antigen interaction, andAG_(linker) is the free energy due to linking each antigen (e.g.,entropy loss, conflicts in geometry, etc.). Since AG=−RT ln (K),

−RT ln(K _(total))=−RT ln(K ₁)−RT ln(K ₂)+RT ln(K _(linker))

If AG_(linker) is negligible (i.e., 0), the binding constants aremultiplicative:

−RT ln(K _(total))=−RT ln(K ₁)−RT ln(K ₂)

−RT ln(K _(total))=−RT[ln(K ₁)+ln(K ₂)]

−RT ln(K _(total))=−RT[ln(K ₁ ·K ₂)]

K _(total) =K ₁ ·K ₂

Assuming proper geometry and minimal entropy loss for the mLCN2-Fcprodrug construct, the affinity between bivalent molecules mLCN2-Fc andCTLA4-Fc mask is 5.5×10⁻⁹ M·5.5×10⁻⁹ M=3.0×10⁻¹⁷ M (see Chapter 2 forK_(D) determination of monomeric mLCN2:CTLA4 interaction). If theprotease substrate on the mask linker is hydrolized, avidity is lost andthe affinity returns to 5.5 nM, thereby permitting dissociation of themask and subsequent activation of mLCN2-Fc.

However, the k_(d) between monovalent mLCN2 and CTLA4 was observed to be2×10⁻⁴ s⁻¹. Using the equation for dissociation half-life time,t_(1/2)=ln(2)/k_(d), we find the dissociative half-life time ofmonomeric CTLA4 and mLCN2 to be 3465 s (˜58 min). Mutations wereintroduced into the CTLA4 mask at the binding interface to hastendissociation upon activation. Once dissociated, the CTLA4 mask couldpotentially bind endogenous ligands CD80 and CD86 and function as animmunosuppressant (e.g., abatacept [2]). Using crystal structures andpublished biochemical reports, we investigated mutations on the CTLA4mask to abrogate binding to CD80/CD86 as well as address thedissociation obstacle [3, 4].

Described herein is the engineering of a non-covalent mask for mLCN2-Fcfor intratumoral delivery of a CTLA4 antagonist. The mask consists ofhuman CTLA4 extracellular domain fused via an MMP9 substrate to IgG1,conferring high affinity through avidity to the system. Cell-stainingand functional assays showed intact masked prodrug was incapable ofbinding CTLA4. Ligand mask, both apo and complexed with mLCN2-Fc, wascleaved by MMP9 in vitro. Upon activation by MMP9—a proteaseoverexpressed in many human tumors—avidity is lost and the dissociationof mask would permit mLCN2-Fc binding to CTLA4 on tumor-infiltrationlymphocytes.

Materials and Methods

Molecular Biology. Human CD86 extracellular domain (UniProt entry:P42081) was cloned to the N-terminus of IgG1 (UniProt entry: P01857) andinserted into the insect cell expression vector pVL1393 with a secretionsignal gp67 at the N-terminus [5, 6]. DNA sequencing confirmedsuccessful subcloning (City of Hope DNA Sequencing Core, Duarte,Calif.).

Protein expression and purification of Fc constructs. Bivalentconstructs containing human Fc (IgG1) were cloned in the pVL1393 vectorand transfected into Sf9 insect cells with BestBac 2.0 BaculovirusCotransfection kit (Expression System). High titer virus was generatedand used to infect Tni cells at an MOI of 3 for protein production.Cells were harvested 48 h post-infection, centrifuged, and supernatantwas applied to Protein A resin (GenScript). Column was washedextensively with PBS, and protein was eluted with 0.1 M glycine pH 3.0and immediately pH neutralized with 1 M Tris-HCl pH 8.0. Concentratedeluate was applied to HiLoad 26/60 Superdex 200 column (GE Healthcare)in PBS and peak fractions were concentrated, flash frozen, and stored at−80° C.

Surface Plasmon Resonance (SPR). SPR experiments were performed on a GEBiacore T100 instrument at 25° C. Ligands were amine coupled to CMSchips at 300_(Rmax). Blank lanes were used as baseline control. Analytetitrations were prepared as serial dilutions in HBS-EP⁺ running buffer(10 mM Hepes pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% v/v P20). Increasingconcentrations of analytes were flowed over blank and ligand lanes at 50μL/min. Regeneration buffer (1 M NaCl, 10 mM glycine pH 2.6) was flowedat 90 μL/min for 10 s. Data analyses were performed using Biacore T100Evaluation Software, version 1.1.1 (GE Healthcare). Response reported asthe difference between blank and peptide lanes.

In vitro MMP9 cleavage assays. Time-course cleavage assay: CTLA4-Fc MMP9and Mxx9 proteins were complexed with mLCN2-Fc and purified bygel-filtration. Samples were dialyzed against cleavage buffer (5 mMCaCl2; 0.02% NP-40, 50 mM Tris pH 7.4, 150 mM NaCl). 8 jig of apo orcomplex samples were treated with 1 jig recombinant human MMP9(ProSpecBio) or equivalent volume buffer and incubated at 37° C. Atselected times, samples were mixed with 2×SDS running buffer in thepresence or absence of DTT and loaded on a 5% stacking/12% resolvingSDS-PAGE. Gel electrophoresis was performed at 150 V for 45 min and gelwas stained with Coomassie Brilliant Blue. Temperature-dependencecleavage assay: 125 jig of CTLA4-Fc MMP9 was incubated 12 hours at 4°,25°, or 37° C. in the presence or absence of 0.2 jig recombinant humanMMP-9. Samples were run in non-reducing conditions on a 8-25% gradientPhastGel (GE) and stained with Coomassie Brilliant Blue.

Flow cytometry. Murine cells: Splenocytes were isolated from C57BL/6mice; 75E6 cells per well were plated in a 6-well plate in IMDM with 10%FBS plus antibiotic/antimycotic. For primed samples, T-cells wereco-cultured with 20% sterile-filtered tumor supernatant (TSN) from C4murine melanoma cell culture for 24 h. Following incubation, denotedsamples received 20 jig CTLA4-Fc MMP9 in PBS immediately chased with 20jig Alexa Fluor 647-conjugated mLCN2-Fc in PBS. Sterile PBS immediatelychased with 20 jig 647̂mLCN2-Fc in PBS was added to remaining treatmentwells. Cells were incubated one hour at 37° C., protected from light.Cells were collected and spun 1500 RPM 4 C for 5 min. Supernatant wasaspirated and cells were resuspended in 450 μL staining buffer (SB, 2%BSA in PBS). Cells were treated with 1:100 dilution of α-FcγR (Robosep)for five minutes. For each experimental group, 200 μL of cells weredistributed to 2 flow cytometry tubes. A 1:50 dilution of eitherα-muCD4-FITC or α-muCD8-FITC (BD Biosciences) was added to sample tubes,subsequently covered and incubated on ice for 30 min protected fromlight. Cells were washed with SB and resuspended in 300 μL DAPIsolution. Samples were analyzed on BD Accuri C6 system (BD Biosciences).Data were analyzed using FlowJo software (Tree Star).

Human cells: Human Daudi cells (ATCC, CCL-213) were maintained inRPMI-1649 medium plus 10% FBS. For flow cytometric experiments, cellswere washed twice with SB. 2E6 cells per sample were stained withindicated amounts of Alexa Fluor 647-conjugated CTLA4-Fc for 30 min inthe dark at room temperature. Cells were washed twice and resuspended in1:200 DAPI in staining buffer. Flow cytometry was performed at the Cityof Hope FACS facility with a CyAn ADP 9 (Beckman Coulter) and the datawere analyzed using FlowJo software (Tree Star). Analytical sizeexclusion chromatography (SEC). Purified samples were mixed 1:1 molarratio, prepared at 6 μM in PBS and incubated for 1 h at 4° C. SEC wasperformed at 4° C. using a Superdex 200 10/300 GL (GE Healthcare) andmonitored at 230 nm. ELISpot IFN-γ ELISpot was performed according tomanufacturer's protocol (eBioscience). Briefly, splenocytes wereharvested from OT-I C57BL/6-Tg(TcraTcrb)1100Mjb/Crl transgenic mice(Charles River). In vitro activation: Single-cells suspensions ofsplenocytes were co-cultured with phytohaemagglutinin (PHA) orirradiated B16-TAC cells (B16 murine melanoma cells expressing TACantigen [7]) in the presence or absence of sterile mLCN2-Fc orpre-complexed mLCN2-Fc:CTLA4-Fc for five days. In vivo activation: OT-Imice were injected at day 0 and 7 with adenovirus encoding TAC antigenas previously described [8]. At day 14 spleens were harvested and mixedwith irradiated B16-TAC cells. Samples from all activation methods wereincubated on anti-IFN-γ antibody-coated ELISpot plate for 24 h. IFN-γproduction was measured by ELISpot plate reader (Cellular Technology,Ltd.).

Production and Purification of CTLA4-Fc MMP9 Mask

A protease-cleavable, non-covalent mask for mLCN2-Fc was created bymodifying the recombinant CTLA4-Fc protein. Specifically, a sequence inthe linker between CTLA4 and Fc was mutated to VPLSLYS, a hydrolyzableMMP9 substrate [9]. This protein mask [CTLA4-Fc MMP9] was biosynthesizedby insect cells at a yield of −20 mg/L growth.

As demonstrated by SEC and SPR, mLCN2-Fc bound to WT CTLA4-Fc(henceforth called “CTLA4-Fc Mxx9,” i.e., non-cleavable mask) with highaffinity. This trend was also seen with MMP9-cleavable CTLA4-Fc MMP9.However, when mLCN2-Fc was mixed at equimolar ratio with CTLA4-Fc MMP9,multiple peaks were seen at a lower elution volume by analytical SECanalysis (FIG. 6). mLCN2-Fc was mixed with CTLA4-Fc at concentrations of20 μM per protein (“fast mixing,” FIG. 6A) and three major peaks ofhigher elution volume were observed: Peak 1 (volumetric range from7.6-8.6 mL) accounted for ˜25% of the area under the curve (AUC); Peak 2(from 8.6-9.6 mL) was 38% of AUC; and Peak 3 (from 10.1-11.1 mL) was 37%of AUC. When lower initial concentrations of mLCN2-Fc and CTLA4-Fc (40nM each protein) were mixed and given time to come to equilibrium priorto SEC analysis, three peaks of higher elution volume were also seen(“slow mixing,” FIG. 6A purple trace). Peak 3 accounted for ˜70% of thetotal AUC, whereas Peaks 1 and 2 combined for the remaining 30%.

The three peaks from each SEC were isolated and separately reanalyzed bySEC. Peak 1 from the both reactions eluted at three separate peaks ofhigher molecular weight (FIGS. 6B and 6C). Peak 2 from both initialreactions eluted as two major peaks (orange). Peak 3, which contained1:1 complexes of mLCN2-Fc and CTLA4-Fc, eluted as one major peak at 10.5mL (grey).

In Vitro MMP9 Cleavage of Non-Covalent Mask

Recombinant CTLA4-Fc MMP9 mask contains MMP9 hydrolysis substrate,VPLSLYS, between CTLA4 and Fc domains. To assess MMP9 cleavage, we alsocreated a mask that was incapable of MMP9 cleavage (CTLA4-Fc Mxx9). Bothmasks were capable of binding mLCN2-Fc, qualitatively shown by SEC. Aseries of in vitro experiments tested the cleavage of CTLA4-Fc MMP9 byrecombinant human MMP9.

Incubating CTLA4-Fc MMP9 with recombinant MMP9 at 37° C. for 9 hresulted in two major protein bands, as resolved by SDS-PAGE (FIG. 3).Addition of MMP9 had no effect on CTLA4-Fc Mxx9 bands. Next, MMP9cleavage reactions were tested at different temperatures to assess thetemperature dependence of CTLA4-Fc MMP9 cleavage. Reactions at 25° C.and 37° C. had similar band intensities of cleavage products, whereas astronger band at the molecular weight of intact mask was seen in thereaction at 4° C. (FIG. 3). A time-course experiment analyzed the rateof in vitro CTLA4-Fc MMP9 cleavage at 37° C. (FIG. 3). At 2 h theintensity of the full-length band was ˜50% of the 0 h timepoint, and at8 h most of the band was gone. Finally the cleavage of pre-complexedmLCN2-Fc:CTLA4-Fc MMP9 was assessed (FIG. 3). Complexed CTLA4-Fc MMP9was cleaved at a rate similar to that of apo CTLA4-Fc MMP9 (FIG. 3).

CTLA4-Fc MMP9 Mask Inhibits mLCN2-Fc Binding to T-Cells

Binding of CTLA4 on live cells was tested in a competition assay bystaining murine T-cells with 647′mLCN2-Fc or CTLA4-Fc MMP9 plus647′mLCN2-Fc. Fluorescently labeled 647′mLCN2-Fc was added to naïve andprimed cells in the presence or absence of CTLA4-Fc MMP9. CD4⁺ and CD8⁺cells were subsequently identified by flow cytometry and analyzed formLCN2-Fc staining (FIG. 7). A 5-fold and 3-fold reduction of mLCN2-Fcstaining in CD4⁺ and CD8⁺ cells, respectively, was seen with theaddition of CTLA4-Fc. In cells treated with mLCN2-Fc only, we observed aslight increase in binding between the naïve and primed T-cell samplesof both CD4⁺ (2.7% increase) and CD8⁺ (14.8%) cells.

Functional assay by cytokine ELISpot analysis tested the immunestimulation of masked mLCN2-Fc. Splenocytes from OT-I transgenic mice,activated with PHA or irradiated B16-TAC cells in vitro or withadenovirus encoding TAC antigen in vivo, were incubated with apomLCN2-Fc or mLCN2-Fc:CTLA4-Fc MMP9 complex for five days. Cells wereapplied to an ELISpot to quantify IFN-γ production (i.e., T-cellstimulation). In all tested settings of T-cell activation, co-incubationwith mLCN2-Fc had significantly higher stimulation of IFN-γ productioncompared to co-incubation with mLCN2-Fc:CTLA4-Fc complex (FIG. 8). Thelargest differential in IFN-γ production was seen in the in vitroactivation by B16-TAC cells experimental groups (FIG. 8B). In thisactivation setting, we observed 163±6 IFN-γ producing cells in samplestreated with apo mLCN2-Fc, whereas samples treated withmLCN2-Fc:CTLA4-Fc generated 21±3 IFN-γ producing cells. For comparison,cells treated with only media yielded 15±7 IFN-γ producing cells.

Non-Covalent Mask Binds Human CD86 In Vitro

The binding of CTLA4-Fc mask to endogenous ligand of CTLA4, CD86, wasanalyzed by flow cytometry and SEC. Fluorescent dye 647 was conjugatedto CTLA4-Fc MMP9 and tested by flow cytometry for binding to Daudicells, a human lymphoma cell line that expresses CD80 and CD86. Weobserved 647″CTLA4-Fc stained Daudi cells in a concentration-dependentmanner (FIG. 9). We also produced and purified recombinant bivalentCD86-Fc (human CD86 extracellular domain fused to human IgG1). Whenmixed 1:1 with CTLA4-Fc and analyzed by SEC, the elution peak signifiesformation of a higher molecular weight complex (FIG. 10).

Further Engineering of the CTLA4-Fc Mask

To reduce the affinity of CTLA4-Fc MMP9 mask for CD80/CD86, we analyzedthe crystal structures of CTLA4 bound to these ligands and to mLCN2.Looking at the superposition of the three crystal structures, there isan overlapping region of CTLA4 that interacts with all three ligands.However, there are also several residues on CTLA4 that contact CD80 andCD86 uniquely (Glu31, Arg33, Thr51) and several residues that recognizeonly mLCN2 (Lys95, Glu97). We made point mutations at these residues onour CTLA4-Fc mask to reduce affinities for CD80 and CD86 as well asincrease the k_(d) between the mask and mLCN2.

When analyzed by SEC, apo CTLA4-Fc variants eluted at 12.2 mL (FIG. 10).CTLA4-Fc variant E31S eluted at 10.4 mL when incubated with CD86-Fc,signifying binding to CD86-Fc. Two elution peaks were noted when E31AR33A was mixed with CD86-Fc, indicating heterogeneous mixture of boundand unbound protein. E31S R33E T51H showed no binding of CD86-Fc, asthis sample eluted as one peak at 12.1 mL.

SEC was also utilized to assess binding of CTLA4-Fc to mLCN2-Fc. E31Seluted as two peaks of higher molecular size with the addition ofmLCN2-Fc (FIG. 11). A major elution peak from T51H mixture was observedat 10.2 mL. These two variants demonstrated substantial binding tomLCN2-Fc. Intermediate peaks of higher molecular sizes were seen forsamples E31A R31A and Y52F, suggesting heterogeneous binding tomLCN2-Fc. E31S R33E T51H eluted at 12.2 mL, signifying no binding tomLCN2-Fc.

All CTLA4-Fc variants tested had lower affinity for mLCN2-Fc compared toCTLA4-Fc WT (Table 3). T51H had a K_(D) of 9.07 nM and a lid of 2.20E-04s⁻¹. Y52F had a K_(D) of 6.14 nM and a lid of 2.15E-04 s⁻¹. E31A R33Ahad a K_(D) of 100 nM and a lid of 1.10E-03 s⁻¹. E31S R33E T51H had aK_(D) of 5600 nM and a k_(d) of 2.50E-02 s⁻¹.

TABLE 3 Kinetics of CTLA4-Fc mask variants, as measured by SPR.Calculated CTLA4-FC Variant k_(a) (M⁻¹ s⁻¹) k_(d) (s⁻¹) K_(D) (M)t_(1/2) (s) WT 3.64E04 2.01E−04 5.52E−09 3466 E31A R33A 1.10E04 1.10E−031.00E−07 693 E31S R33E T51H 4.43E03 2.50E−02 5.60E−06 28 T51H 2.42E042.20E−04 9.07E−09 3151 Y52F 3.51E04 2.15E−04 6.14E−09 3224 Bindingkinetics of mLCN2 for each CTLA4-Fc variant was measured by SPR.Half-life time was calculated from equation: t_(1/2) = ln (2)/k_(d).

REFERENCES

-   1. Donaldson, J. M., et al., Design and development of masked    therapeutic antibodies to limit off-target effects: application to    anti-EGFR antibodies. Cancer Biol Ther, 2009. 8(22): p. 2147-52.-   2. Maxwell, L. J. and J. A. Singh, Abatacept for rheumatoid    arthritis: a Cochrane systematic review. J Rheumatol. 37(2): p.    234-45.-   3. Morton, P. A., et al., Differential effects of CTLA-4    substitutions on the binding of human CD80 (B7-1) and CD86 (B7-2). J    Immunol, 1996. 156(3): p. 1047-54.-   4. Xu, Z., et al., Affinity and cross-reactivity engineering of    CTLA4-Ig to modulate T cell costimulation. J Immunol. 189(9): p.    4470-7.-   5. Charles, I. G., et al., Cloning and expression of a rat neuronal    nitric oxide synthase coding sequence in a baculovirus/insect cell    system. Biochem Biophys Res Commun, 1993. 196(3): p. 1481-9.-   6. Au, L. C., et al., Secretory production of bioactive recombinant    human granulocyte-macrophage colony-stimulating factor by a    baculovirus expression system. J Biotechnol, 1996. 51(2): p. 107-13.-   7. Waldman, S. A., Annual Progress Report: 2009 Nonformula Grant on    Cancer Vaccines. 2011, Thomas Jefferson University: Philadelphia,    Pa. p. 1-9.-   8. Igoucheva, O., et al., Immunotargeting and eradication of    orthotopic melanoma using a chemokine-enhanced DNA vaccine. Gene    Ther, 2013. 20(9): p. 939-48.-   9. Turk, B. E., et al., Determination of protease cleavage site    motifs using mixture-based oriented peptide libraries. Nat    Biotechnol, 2001. 19(7): p. 661-7.

VII. EMBODIMENTS Embodiment 1

A recombinant masking protein comprising two identical masking proteindomains, each of said masking protein domains comprising: (i) a maskingdimerizing domain; (ii) a ligand-masking binding domain; and (iii) acleavable masking linker connecting said ligand-masking binding domainto said masking dimerizing domain, wherein said masking protein domainsare bound together.

Embodiment 2

The recombinant masking protein of embodiment 1, wherein said maskingdimerizing domain is an Fc protein domain.

Embodiment 3

The recombinant masking protein of embodiment 2, wherein said Fc proteindomain is an IgG₁ Fc protein.

Embodiment 4

The recombinant masking protein of any one of embodiments 1-3, whereinsaid ligand-masking binding domain is a small molecule or a cellularprotein domain.

Embodiment 5

The recombinant masking protein of embodiment 4, wherein said cellularprotein domain is a cellular growth factor domain, a cellular surfaceprotein domain or functional fragment thereof.

Embodiment 6

The recombinant masking protein of embodiment 5, wherein said cellulargrowth factor domain is a TNF domain.

Embodiment 7

The recombinant masking protein of embodiment 5, wherein said cellularsurface protein domain is an Erbb receptor domain or a T cell receptordomain.

Embodiment 8

The recombinant masking protein of embodiment 7, wherein said Erbbreceptor domain is a Her2 domain or EGFR domain.

Embodiment 9

The recombinant masking protein of embodiment 7, wherein said T cellreceptor domain is a CTLA-4 domain.

Embodiment 10

The recombinant masking protein of one of embodiments 1-9, wherein saidcleavable masking linker comprises a protease cleavage site.

Embodiment 11

The recombinant masking protein of embodiment 10, wherein said proteasecleavage site is a matrix metalloprotease cleavage site, a disintegrinand metalloproteinase domain-containing (ADAM) metalloprotease cleavagesite or a prostate specific antigen (PSA) protease cleavage site.

Embodiment 12

The recombinant masking protein of one of embodiments 1-11, wherein saidrecombinant masking protein is bound to a recombinant ligand proteincomprising two identical ligand protein domains, each of said ligandprotein domains comprising: (i) a ligand dimerizing domain; (ii) aligand domain; bound to one of said ligand-masking domains; and (iii) aligand linker connecting said ligand domain to said ligand dimerizingdomain, wherein said ligand protein domains are bound together.

Embodiment 13

The recombinant masking protein of embodiment 12, wherein said liganddimerizing domain is an Fc protein domain.

Embodiment 14

The recombinant masking protein of embodiment 13, wherein said Fcprotein domain is an IgG₁ Fc protein.

Embodiment 15

The recombinant masking protein of one of embodiments 12-14, whereinsaid ligand domain is a cellular protein binding domain.

Embodiment 16

The recombinant masking protein of embodiment 15, wherein said cellularprotein binding domain is a cellular growth factor binding domain or acellular surface protein binding domain.

Embodiment 17

The recombinant masking protein of embodiment 16, wherein said cellulargrowth factor binding domain is a TNF receptor domain.

Embodiment 18

The recombinant masking protein of embodiment 16, wherein said cellularsurface protein binding domain is an Erbb receptor binding domain or a Tcell receptor binding domain.

Embodiment 19

The recombinant masking protein of embodiment 18, wherein said Erbbreceptor binding domain is a Her2 binding domain or an EGFR bindingdomain.

Embodiment 20

The recombinant masking protein of embodiment 18, wherein said T cellreceptor binding domain is a CTLA-4 binding domain.

Embodiment 21

The recombinant masking protein of embodiment 20, wherein said CTLA-4binding domain is LCN2.

Embodiment 22

The recombinant masking protein of one of embodiments 12-21, whereinsaid ligand domain comprises a CDR domain.

Embodiment 23

The recombinant masking protein of one of embodiments 12-22, whereinsaid ligand domain is an antibody domain.

Embodiment 24

The recombinant masking protein of one of embodiments 1-23, wherein saidmasking-dimerizing domains connect to a targeting domain through atargeting linker.

Embodiment 25

The recombinant masking protein of embodiment 24, wherein said targetingdomain is a single-chain variable fragment (scFv) domain.

Embodiment 26

The recombinant masking protein of embodiment 25, wherein said scFvdomain is a hemaglutinin (HA) scFv domain.

Embodiment 27

The recombinant masking protein of embodiment 24, wherein said targetinglinker is connected to the C-terminus of said masking dimerizing domain.

Embodiment 28

The recombinant masking protein of embodiment 27, wherein said cleavablemasking linker is connected to the N-terminus of said masking dimerizingdomain.

Embodiment 29

The recombinant masking protein of one of embodiments 24-28, whereinsaid targeting linker is a cleavable targeting linker.

Embodiment 30

The recombinant masking protein of embodiment 29, wherein said cleavabletargeting linker comprises a protease cleavage site.

Embodiment 31

The recombinant masking protein of embodiment 30, wherein said proteaseis a matrix metalloprotease cleavage site, a disintegrin andmetalloproteinase domain-containing (ADAM) metalloprotease cleavage siteor a prostate specific antigen (PSA) protease cleavage site.

Embodiment 32

The recombinant masking protein of one of embodiments 12-31, whereinsaid ligand dimerizing domains connect to a targeting domain through atargeting linker.

Embodiment 33

The recombinant masking protein of embodiment 32, wherein said targetingdomain is a single-chain variable fragment (scFv) domain.

Embodiment 34

The recombinant masking protein of embodiment 33, wherein said scFvdomain is a hemaglutinin (HA) scFv domain.

Embodiment 35

The recombinant masking protein of embodiment 32, wherein said targetinglinker is connected to the C-terminus of said ligand dimerizing domain.

Embodiment 36

The recombinant masking protein of embodiment 32, wherein said ligandlinker is connected to the N-terminus of said ligand dimerizing domain.

Embodiment 37

The recombinant masking protein of embodiment 32, wherein said targetinglinker is a cleavable targeting linker.

Embodiment 38

The recombinant masking protein of embodiment 37, wherein said cleavabletargeting linker comprises a protease cleavage site.

Embodiment 39

The recombinant masking protein of embodiment 38, wherein said proteasecleavage site is a matrix metalloprotease cleavage site, a disintegrinand metalloproteinase domain-containing (ADAM) metalloprotease cleavagesite or a prostate specific antigen (PSA) protease cleavage site.

Embodiment 40

A method of treating a disease in a subject in need thereof, said methodcomprising administering to a subject a therapeutically effective amountof a recombinant masking protein comprising two identical maskingprotein domains, each of said masking protein domains comprising: (i) amasking dimerizing domain; (ii) a ligand-masking binding domain; and(iii) a cleavable masking linker connecting said ligand-masking bindingdomain to said masking dimerizing domain, wherein said masking proteindomains are bound together; and a recombinant ligand protein comprisingtwo identical ligand protein domains, each of said ligand proteindomains comprising: (i) a ligand dimerizing domain; (ii) a liganddomain; and (iii) a ligand linker connecting said ligand domain to saidligand dimerizing domain, wherein said ligand protein domains are boundtogether.

Embodiment 41

The method of embodiment 40, wherein said recombinant masking proteinand said recombinant ligand protein are administered to said subjectsimultaneously.

Embodiment 42

The method of embodiment 40, wherein said ligand domain is bound to atleast one of said ligand-masking domains prior to said administering.

Embodiment 43

The method of embodiment 40, wherein said masking-dimerizing domainsconnect to a targeting domain through a targeting linker.

Embodiment 44

The method of embodiment 43, wherein said targeting linker is connectedto the C-terminus of said masking dimerizing domain.

Embodiment 45

The method of embodiment 44, wherein said cleavable masking linker isconnected to the N-terminus of said masking dimerizing domain.

Embodiment 46

The method of embodiment 43, wherein said targeting linker is acleavable targeting linker.

Embodiment 47

The method of embodiment 40, wherein said ligand dimerizing domainsconnect to a targeting domain through a targeting linker.

Embodiment 48

The method of embodiment 47, wherein said targeting linker is connectedto the C-terminus of said ligand dimerizing domain.

Embodiment 49

The method of embodiment 48, wherein said ligand linker is connected tothe N-terminus of said ligand dimerizing domain.

Embodiment 50

The method of embodiment 47, wherein said targeting linker is acleavable targeting linker.

Embodiment 51

The method of embodiment 40, wherein said disease is cancer, anautoimmune disease, a neurodegenerative disease or a cardiovasculardisease.

Embodiment 52

The method of embodiment 51, wherein said cancer is leukemia, lymphoma,head and neck cancer, colorectal cancer, prostate cancer, melanoma,breast cancer or neuroblastoma.

Embodiment 53

The method of embodiment 51, wherein said autoimmune disease is Crohn'sdisease, rheumatoid arthritis, asthma or psoriasis.

Embodiment 54

The method of embodiment 51, wherein said neurodegenerative disease isAlzheimer's Disease or multiple sclerosis.

Embodiment 55

A pharmaceutical composition comprising a pharmaceutically acceptableexcipient and a recombinant masking protein comprising two identicalmasking protein domains, each of said masking protein domainscomprising: (i) a masking dimerizing domain; (ii) a ligand-maskingbinding domain; and (iii) a cleavable masking linker connecting saidligand-masking binding domain to said masking dimerizing domain, whereinsaid masking protein domains are bound together; and a recombinantligand protein comprising two identical ligand protein domains, each ofsaid ligand protein domains comprising: (i) a ligand dimerizing domain;(ii) a ligand domain; and (iii) a ligand linker connecting said liganddomain to said ligand dimerizing domain, wherein said ligand proteindomains are bound together.

Embodiment 56

The pharmaceutical composition of embodiment 55, wherein said liganddomain is bound to one of said ligand-masking domains.

Embodiment 57

The pharmaceutical composition of embodiment 55, wherein saidmasking-dimerizing domains connect to a targeting domain through atargeting linker.

Embodiment 58

The pharmaceutical composition of embodiment 57, wherein said targetinglinker is connected to the C-terminus of said masking dimerizing domain.

Embodiment 59

The pharmaceutical composition of embodiment 58, wherein said cleavablemasking linker is connected to the N-terminus of said masking dimerizingdomain.

Embodiment 60

The pharmaceutical composition of embodiment 57, wherein said targetinglinker is a cleavable targeting linker.

Embodiment 61

The pharmaceutical composition of embodiment 55, wherein said liganddimerizing domains connect to a targeting domain through a targetinglinker.

Embodiment 62

The pharmaceutical composition of embodiment 61, wherein said targetinglinker is connected to the C-terminus of said ligand dimerizing domain.

Embodiment 63

The pharmaceutical composition of embodiment 62, wherein said ligandlinker is connected to the N-terminus of said ligand dimerizing domain.

Embodiment 64

The pharmaceutical composition of embodiment 61, wherein said targetinglinker is a cleavable targeting linker.

Embodiment 65

A kit comprising: (A) a recombinant masking protein comprising twoidentical masking protein domains, each of said masking protein domainscomprising: (i) a masking dimerizing domain; (ii) a ligand-maskingbinding domain; and (iii) a cleavable masking linker connecting saidligand-masking binding domain to said masking dimerizing domain, whereinsaid masking protein domains are bound together; and (B) a recombinantligand protein comprising two identical ligand protein domains, each ofsaid ligand protein domains comprising: (i) a ligand dimerizing domain;(ii) a ligand domain; and (iii) a ligand linker connecting said liganddomain to said ligand dimerizing domain, wherein said ligand proteindomains are bound together.

Embodiment 66

The kit of embodiment 65, wherein said recombinant masking protein andsaid recombinant ligand protein are in one container.

Embodiment 67

The kit of embodiment 66, wherein said ligand domain is bound to one ofsaid ligand-masking domains.

Embodiment 68

The kit of embodiment 65, wherein said recombinant masking protein andsaid recombinant ligand protein are in separate containers.

1. A recombinant masking protein comprising: (i) a masking dimerizingdomain, wherein said masking dimerizing domain is an Fc protein domain;(ii) a ligand-masking binding domain; and (iii) a cleavable maskinglinker covalently bound to said ligand-masking binding domain and tosaid masking dimerizing domain.
 2. (canceled)
 3. The recombinant maskingprotein of claim 1, wherein said Fc protein domain is an IgG₁ Fcprotein.
 4. The recombinant masking protein of claim 1, wherein saidligand-masking binding domain is a small molecule, a peptide, or acellular protein domain.
 5. The recombinant masking protein of claim 4,wherein said cellular protein domain is a cellular growth factor domain,a cellular surface protein domain or functional fragment thereof.
 6. Therecombinant masking protein of claim 5, wherein said cellular growthfactor domain is a TNF domain.
 7. The recombinant masking protein ofclaim 5, wherein said cellular surface protein domain is an Erbbreceptor domain or a T cell receptor domain.
 8. The recombinant maskingprotein of claim 7, wherein said Erbb receptor domain is a Her2 domainor EGFR domain.
 9. The recombinant masking protein of claim 7, whereinsaid T cell receptor domain is a CTLA-4 domain.
 10. The recombinantmasking protein of claim 1, wherein said cleavable masking linkercomprises a protease cleavage site.
 11. The recombinant masking proteinof claim 10, wherein said protease cleavage site is a matrixmetalloprotease cleavage site, a disintegrin and metalloproteinasedomain-containing (ADAM) metalloprotease cleavage site or a prostatespecific antigen (PSA) protease cleavage site.
 12. The recombinantmasking protein of claim 1, wherein said recombinant masking protein isbound to a recombinant ligand protein, comprising: (i) a liganddimerizing domain; (ii) a ligand domain; bound to one of saidligand-masking domains; and (iii) a ligand linker connecting said liganddomain to said ligand dimerizing domain.
 13. The recombinant maskingprotein of claim 12, wherein said ligand dimerizing domain is an Fcprotein domain.
 14. The recombinant masking protein of claim 13, whereinsaid Fc protein domain is an IgG₁ Fc protein.
 15. The recombinantmasking protein of claim 12, wherein said ligand domain is a cellularprotein binding domain.
 16. The recombinant masking protein of claim 15,wherein said cellular protein binding domain is a cellular growth factorbinding domain or a cellular surface protein binding domain.
 17. Therecombinant masking protein of claim 16, wherein said cellular growthfactor binding domain is a TNF receptor domain.
 18. The recombinantmasking protein of claim 16, wherein said cellular surface proteinbinding domain is an Erbb receptor binding domain or a T cell receptorbinding domain.
 19. The recombinant masking protein of claim 18, whereinsaid Erbb receptor binding domain is a Her2 binding domain or an EGFRbinding domain.
 20. The recombinant masking protein of claim 18, whereinsaid T cell receptor binding domain is a CTLA-4 binding domain.
 21. Therecombinant masking protein of claim 20, wherein said CTLA-4 bindingdomain is LCN2.
 22. The recombinant masking protein of claim 12, whereinsaid ligand domain comprises a CDR domain.
 23. The recombinant maskingprotein of claim 22, wherein said ligand domain is an antibody domain.24-68. (canceled)